Trastuzumab Triggers Phagocytic Killing of High HER2 Cancer Cells In Vitro and In Vivo by Interaction with Fcγ Receptors on Macrophages

Yun Shi, Xuejun Fan, Hui Deng, Randall J. Brezski, Michael Rycyzyn, Robert E. Jordan, William R. Strohl, Quanming Zou, Ningyan Zhang and Zhiqiang An
J Immunol May 1, 2015, 194 (9) 4379-4386; DOI: https://doi.org/10.4049/jimmunol.1402891

Abstract

Trastuzumab has been used for the treatment of HER2-overexpressing breast cancer for more than a decade, but the mechanisms of action for the therapy are still being actively investigated. Ab-dependent cell-mediated cytotoxicity mediated by NK cells is well recognized as one of the key mechanisms of action for trastuzumab, but trastuzumab-mediated Ab-dependent cellular phagocytosis (ADCP) has not been established. In this study, we demonstrate that macrophages, by way of phagocytic engulfment, can mediate ADCP and cancer cell killing in the presence of trastuzumab. Increased infiltration of macrophages in the tumor tissue was associated with enhanced efficacy of trastuzumab whereas depletion of macrophages resulted in reduced antitumor efficacy in mouse xenograft tumor models. Among the four mouse FcγRs, FcγRIV exhibits the strongest binding affinity to trastuzumab. Knockdown of FcγRIV in mouse macrophages reduced cancer cell killing and ADCP activity triggered by trastuzumab. Consistently, an upregulation of FcγRIV expression by IFN-γ triggered an increased ADCP activity by trastuzumab. In an analogous fashion, IFN-γ priming of human macrophages increased the expression of FcγRIII, the ortholog of murine FcγRIV, and increased trastuzumab-mediated cancer cell killing. Thus, in two independent systems, the results indicated that activation of macrophages in combination with trastuzumab can serve as a therapeutic strategy for treating high HER2 breast cancer by boosting ADCP killing of cancer cells.

Introduction

Trastuzumab is a humanized mAb for the treatment of HER2-overexpressing breast cancer (1). After >15 years of successful clinical use, the mechanisms of action for trastuzumab are still being investigated. Among the established modes of action for trastuzumab are inhibition of HER2-mediated cell signaling (2–4) and Ab-dependent cell-mediated cytotoxicity (ADCC) (5). Our group demonstrated recently that engagement of immune cells that express FcγRs with trastuzumab-coated cancer cells mediated HER2 downregulation in the target cells (6).

NK cells play an important role in ADCC, and an increased infiltration of NK cells within breast tumors has been linked to the efficacy of trastuzumab (5). Additionally, macrophages participate in both innate and acquired immunity in cancer. The roles of tumor-associated macrophages (TAMs) are often associated with the promotion of tumor progression and metastasis (7, 8). TAMs still retain Fc-dependent antitumor function despite promoting tumor invasion (8). A study showed that increased TAM infiltration correlated with a positive prognosis in follicular lymphoma patients undergoing rituximab treatment (9). Consistent with this observation is that macrophages are essential for the Ab-dependent depletion of cancer cells mediated by anti-CD20 (10), anti-CD30 (11), and anti-CD40 (12) mAbs in preclinical studies. Macrophages express FcγRs that interact with the Fc portion of IgG Abs (13). Ab-dependent cellular phagocytosis (ADCP) mediated by activated macrophages can kill tumor cells (14). It has been reported that trastuzumab could mediate ADCP against HER2-expressing cancer cells by PBMCs (15) and macrophages (16) in vitro. However, the role of macrophages in response to trastuzumab in antitumor efficacy has not been established in vivo. With this context, a study to clarify the role of macrophages and ADCP in trastuzumab-mediated antitumor efficacy was undertaken.

In this study, we investigated the role of macrophages and ADCP for trastuzumab antitumor efficacy using both in vitro coculturing of macrophages with cancer cells as well as in vivo mouse xenograft tumor models. We demonstrated that FcγRIV expressed on mouse macrophages interact with the Fc domains of cell-bound trastuzumab and thereby induced ADCP-mediated cancer cell killing. The results from this study not only validated ADCP as a new mechanism of action for trastuzumab, but also suggested that the activation of macrophages can improve the anticancer efficacy of trastuzumab and other Ab immune therapies by boosting ADCP.

Materials and Methods

Cell lines and reagents

Cell lines BT474 (human breast cancer cell line), SKOV-3 (human ovarian carcinoma cells), RAW264.7 (mouse macrophage cell line), THP-1 (human monocyte cell line), and HEK 293T cells (a human embryonic kidney 293T cell line) were obtained from American Tissue Culture Collection (Manassas, VA) and cultured in American Tissue Culture Collection–recommended conditions. The L929 cell line (murine aneuploid fibrosarcoma cell line) was from European Collection of Cell Cultures (Sigma-Aldrich, St. Louis, MO). IFN-γ and IL-4 were from ProSpec TechnoGene (Rehovot, Israel). LPS was from Sigma-Aldrich. Cell culture media RPMI 1640 was from Thermo Fisher Scientific (Pittsburgh, PA). FCS was from Invitrogen (Carlsbad, CA).

Generation of mouse bone marrow–derived macrophages and stimulation of different types of macrophages in vitro

Bone marrow–derived macrophages (BMMs) were differentiated from mouse bone marrow cells as reported (17). Briefly, the isolated bone marrow cells were cultured in complete RPMI 1640 medium supplemented with 10% FCS and 30% pretested conditioned medium from the L929 cell line as a source of M-CSF for 5 d (17). BMMs were >95% CD11b+ as measured by flow cytometry. The BMMs were further polarized to type 1 macrophage (M1) by IFN-γ (50 ng/ml) and LPS (10 ng/ml), and to type 2 macrophage (M2) by IL-4 (20 ng/ml). Conditioned medium (cm) from 3-d BT474 cancer cell culture was used to polarize BMMs to Mcm.

Cancer cell killing assay

Cancer cell killing was monitored continuously and noninvasively using the xCELLigence instrument (Roche, Mannheim, Germany) as described previously (18). Briefly, high HER2-expressing SKOV-3 cancer cells were seeded in E-plate 96 (ACEA Biosciences, San Diego, CA), followed by the addition of macrophages (BMMs or RAW264.7 cells) as immune effector cells in the presence of trastuzumab or isotype IgG control (5 μg/ml). Cells alone were used as baseline growth index control. Three wells were used for each treatment group in a 96-well plate assay, and average of the three wells was used as cell growth index of the treatment. Cell growth (measured as cell index) was monitored continuously for 2 d. The E:T ratio was 5:1 or 2:1. The cell index was normalized when the effector cells were added. The normalized cell index recorded after 24 or 48 h of trastuzumab treatment was used to calculate the percentage of cancer cell lysis using the formula: [(cell index of the macrophage group − cell index of the macrophage plus trastuzumab group)/cell index of the macrophage group] × 100. The results were expressed as mean ± SD. Experiments were repeated three times and each treatment contained three replicates.

ADCP assay using flow cytometry and confocal fluorescence microscopy

For the two-color flow cytometry ADCP assay, high HER2-expressing BT474 breast cancer cells were labeled with cell proliferation dye eFluor 670 (eBioscience, San Diego, CA) according to the manufacturer’s instructions. The labeled cells were then incubated with macrophages at a 1:1 ratio with 5 μg/ml trastuzumab or isotype IgG control in RPMI 1640/10% FBS. At the end of 1 h of incubation at 37°C, cells were stained with FITC–anti-CD11b (BD Biosciences, San Jose, CA) for 30 min at 4°C and analyzed by flow cytometry on a guava easyCyte HT instrument (Millipore, Danvers, MA). Cells were gated for CD11b+ cells (macrophages), and the CD11b+ cells were then grouped as single-positive macrophages alone (eFluor 670−, CD11b+) and two-color stained phagocytosed macrophages (eFluor 670+, CD11b+). The percentage of phagocytosis was calculated as the population of phagocytized macrophages among the total macrophages. All tests were performed in triplicate and the results were expressed as mean ± SD. For the ADCP assay by fluorescence microscopy, macrophages were cultured on eight-well chamber slides overnight and eFluor 670–labeled BT474 cells were added to the chamber slides in the presence of trastuzumab (5 μg/ml) or isotype IgG. After 1 h of incubation at 37°C, the media were removed and the slides were washed with 1× PBS. The slides were then stained with FITC–anti-CD11b for 30 min at 4°C and examined under a Carl Zeiss fluorescence microscope (Carl Zeiss, Thornwood, NY). The phagocytized cancer cells in red were localized in the green color–stained macrophages.

Mouse xenograft tumor model and macrophage depletion in vivo

Mouse xenograft studies were carried out in accordance with animal care and use guidelines, and the protocol was approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston. Athymic nu/nu mice (Charles River Laboratories, Wilmington, MA) were used for experiment at age of 7–8 wk. BT474 cells (5 × 106) were implanted s.c. Trastuzumab and isotype IgG were administered once weekly at 5 mg/kg i.p. when tumor size reached 100 mm3. For the macrophage depletion assay, macrophages were depleted by i.p. administration of 200 μl clodrosome (5 mg/ml; Encapsula NanoSciences, Brentwood, TN) weekly as reported (19). Tumor size was measured twice weekly using a Vernier scale caliper. For antitumor efficacy study, mice were treated with six weekly injections of trastuzumab or control isotype IgG. For ex vivo isolation of tumor-infiltrating immune cells, tumors were harvested 3 d after the second Ab injection. Tumor tissue was digested using a tumor dissociation kit (Miltenyi Biotec, Auburn, CA) and homogenates to single-cell suspensions using the gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer’s protocol.

Measurement of FcγR expression and FcγRs binding assays

Macrophages were stained with rabbit anti-mouse FcγRIV (Creative Biomart, Shirley, NY) and followed by Alexa Fluor 488–anti-rabbit IgG (Life Technologies). After staining, the cells were detected using the guava easyCyte HT instrument. Binding constant (Kd) of trastuzumab to human FcγRIII in comparison with murine FcγRIV was determined using the surface plasmon resonance method with a Biacore T-100 instrument as we described previously (20). Binding of mouse FcγRs with trastuzumab was also determined by ELISA adapted from a reported method (18), and FcγR proteins were from Invitrogen. Briefly, FcγR proteins (2 μg/ml) were coated on a MaxiSorp plate (eBioscience), and binding of trastuzumab was detected using an F(ab′)2 fragment of goat anti-human F(ab′)2 Ab conjugated with alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) using the 4-methylumbelliferyl phosphate substrate (Sigma-Aldrich). Fluorescence intensity was read using a plate reader (Molecular Devices, Sunnyvale, CA). FcγRIV expression in ex vivo xenograft tumor tissues was examined by tissue immunofluorescence staining. Briefly, xenograft tumor tissues were excised freshly and snap-frozen in OCT solution (Sigma-Aldrich) and frozen slides were made for immunofluorescence staining. The tumor tissue was stained with rabbit anti-mouse FcγRIV Ab and followed by Alexa Fluor 488–anti-rabbit IgG. The expression of FcγRIV was examined using a Carl Zeiss fluorescence microscope.

Knockdown of FcγRIV in RAW264.7 cells

The RNAi Consortium lentiviral mouse FcγRIV short hairpin RNA (shRNA) was ordered from Thermo Scientific (Fremont, CA). To package the lentivirus, HEK 293T cells were cotransfected by FcγRIV shRNA plasmid psPAX2 and PMD2.G envelope plasmid using 293fectin transfection reagent (Invitrogen) according to the manufacturer’s instructions. At 48 h, the virus was collected to infect the RAW264.7 cells in the presence of Polybrene (4 μg/ml; Sigma-Aldrich). Three days after infection, the cells were selected under puromycin (Sigma-Aldrich). The selected cells were amplified in the presence of puromycin, and FcγRIV expression knockdown was confirmed by flow cytometry.

Statistical analysis

Where appropriate, an unpaired t test between two treatment groups was performed using the GraphPad Prism (version 5) software. A p value < 0.05 between treatment groups was considered significantly different.

Results

Macrophages induced trastuzumab-dependent cancer cell killing

We tested whether trastuzumab can mediate cancer cell killing using BMMs or a murine macrophage cell line (RAW264.7 cells) as effector cells and the high HER2 expression SKOV-3 cells as target cells. Cancer cell lysis mediated by macrophages in the presence and absence of trastuzumab was monitored in real-time and noninvasively using the xCELLigence instrument. The ratio of macrophages (effector cells) and cancer cells (target cells) was 2:1 or 5:1. The cell index was normalized at the point of trastuzumab and macrophage addition (horizontal open arrows in Fig. 1A). As shown in Fig. 1A, the normalized cell index in the presence of trastuzumab was markedly reduced compared with the cells treated with an isotype control IgG shortly after the addition of BMMs; however, the cell growth index with trastuzumab in the absence of BMMs was similar to that of cancer cells alone. In comparison with the cell indices in the isotype control Ab group, the killing of cancer cells by BMMs in the presence of trastuzumab reached a maximum of 40% at the E:T ratio of 2:1, and >60% when the E:T ratio was increased to 5:1 after 24 h of coculture of cancer cells and macrophages (Fig. 1B). The same trend was also observed using the mouse macrophage RAW264.7 cells (Fig. 1C). In comparison with the cell indexes in the isotype control Ab group, the killing of cancer cells by RAW264.7 cells in the presence of trastuzumab was ∼20% at the E:T ratio of 2:1, and >30% when the E:T ratio was increased to 5:1 (Fig. 1D).

FIGURE 1.
FIGURE 1.

Macrophage-induced killing of high HER2–expressing cancer cells in the presence of trastuzumab. SKOV-3 cancer cells were seeded in E-Plate 96, followed by the addition of BMMs (A and B) or RAW264.7 cells (C and D) as immune effector cells (pointed by the horizontal open arrows) in the presence of trastuzumab or isotype IgG control. Cancer cells plus isotype IgG was used as controls. The cell indexes were monitored in real-time for 80 h to map the cell growth curve. The cell index at 24 h after trastuzumab treatment (indicated by the vertical solid arrows in the kinetic graphs) was used to calculate the percentage of cell lysis as shown in (B) and (D). The percentage of macrophage-mediated cancer cell lysis in response to trastuzumab was calculated using the formula: [(cell index with isotype control − cell index of trastuzumab treatment)/cell index of isotype control] × 100. The ratio of macrophage to cancer cells in the coculture was at 2:1 or 5:1 as indicated in the graphs. The experiments were repeated three times. The error bars in (B) and (D) indicate the SD among three independent experiments.

Trastuzumab-opsonized high HER2–expressing cancer cells underwent phagocytosis by macrophages

To determine whether ADCP played key role in cancer cell killing by trastuzumab in the presence of macrophages, we investigated macrophage-mediated phagocytosis in the presence of trastuzumab using both flow cytometry and confocal fluorescence imaging methods. Fluorescent dye eFluor 670–labeled high HER2–expressing BT474 breast cancer cells were used as target cells and incubated with BMMs or RAW264.7 cells as effector cells at an E:T ratio of 1:1 in the presence of trastuzumab or an isotype IgG control. Before detection of phagocytosis of the eFluor 670–prelabeled BT474 cancer cells, macrophages were stained with CD11b-FITC at 4°C and cells with dual colors (red for cancer cells and green for macrophages) were detected by flow cytometry or confocal fluorescence imaging. To measure the population of macrophages with cancer cell phagocytosis, we first gated CD11b+ cells and then analyzed the percentage of macrophages with double staining. As shown in Fig. 2A, a significant increase of the CD11b+/eFlour 670+ cell population was observed in the trastuzumab-treated groups when compared with the isotype IgG control group, suggesting that BT474 cancer cells were phagocytized by BMMs or RAW264.7 cells. To confirm that the double-positive cells detected by flow cytometry were a true representation of phagocytized cancer cells in macrophages, we imaged the phagocytized cells using a confocal fluorescence microscope. In the presence of trastuzumab, the red BT474 cells were clearly visible within the green-labeled macrophage cells (Fig. 2B), whereas little phagocytosis of cancer cells by macrophages was obeserved in the isotype IgG control group. To investigate whether a different activation status of macrophages impacts the phagocytotic killing of Ab opsonized cancer cells, we derived macrophages into type 1 (M1) and type 2 (M2) as well as those derived with cancer cell–conditioned medium (Mcm). The M1 macrophages exhibited the most potent phagocytosis of high HER2 cancer cells in the presence of trastuzumab among the tested groups, and both BMMs and M1 macrophages showed significantly more phagocytosis in the presence of trastuzumab than did that of the isotype Ab control (Fig. 2C). However, M2 and Mcm macrophages had no significant increase of ADCP activities in the presence of trastuzumab in comparison with the presence of isotype control Ab (Fig. 2C), suggesting that activation status of macrophages has an impact on ADCP activity.

FIGURE 2.
FIGURE 2.

Trastuzumab-mediated phagocytosis of cancer cells by macrophages. (A) eFluor 670–labeled BT474 cells were incubated with BMMs (upper panel) or RAW264.7 cells (lower panel) at a 1:1 ratio and treated with trastuzumab or isotype IgG control (5 μg/ml). After 1 h incubation in a cell culture incubator at 5% CO2 and 37°C, cells were stained with FITC–anti-CD11b Ab and analyzed by flow cytometry. A representative flow dot plot is shown for each condition. The cells were gated on CD11b+ cells and the double-positive population (indicating phagocytized cancer cells by macrophage) is boxed in the flow dot plot and quantified in the bar graphs on the right (n = 3). SDs are indicated by the arrow bars. *p < 0.05. (B) Phagocytosis mediated by BMMs (upper panel) or RAW264.7 cells (lower panel) in the presence of trastuzumab was detected using eight-well glass slides and the images were captured with a fluorescent microscope. Original magnification ×40. The green indicates macrophage and the red indicates cancer cells. (C) Phagocytosis (%) by different types of macrophages in the presence of trastuzumab or isotype IgG. SDs are indicated by the arrow bars. The experiments were repeated three times independently. *p < 0.05.

The antitumor efficacy of trastuzumab is correlated with the infiltration of macrophages within tumor sites in a mouse xenograft tumor model

To study the role of macrophages in regard to trastuzumab efficacy in vivo, tumor-infiltrated macrophages were determined following administration of Ab at 5 mg/kg for 2 weekly dosings. The percentage of infiltrated macrophages (CD11b+) was found to be significantly higher in tumor tissues in mice treated with trastuzumab (20.88%) compared with the tumors in control IgG-treated mice (11.47%) (Fig. 3A, bar graph). To study whether the increased infiltration of macrophages in the trastuzumab-treated xenograft tumors was a reflection of a general immune amplification in the treated mice, we measured peripheral immune cell populations isolated from spleen tissues of the treated mice. CD11b+ cells in the spleens from mice treated with trastuzumab (13.69%) were significantly higher than in those treated with the control isotype IgG (5.24%) (Fig. 3A). As expected, trastuzumab exhibited strong inhibition of BT474 breast cancer xenograft tumor growth (Fig. 3B). To further investigate the potential role of macrophages for the antitumor efficacy of trastuzumab, we depleted macrophages in vivo using clodrosome nanoparticles. Tumor-bearing mice were administered clodrosome by i.p. injection 1 d before trastuzumab treatment, and the regimen was repeated weekly for 4 wk. Macrophages in the spleens and tumor sites were isolated from fresh tissues obtained 24 h after the last Ab treatments and analyzed by flow cytometry. Macrophages (CD11b+) were minimally detectable in the spleen and tumor tissues in mice treated with clodrosome in either the trastuzumab or isotype IgG treatment groups (Fig. 3A). Strikingly, the inhibition of tumor growth by trastuzumab was significantly impaired in mice treated with clodrosome in comparison with that without the depletion of macrophages (Fig. 3B). Clodrosome depletion of macrophages did not impact the tumor growth in the isotype control IgG treatment group (Fig. 3B), indicating that clodrosome depletion alone had no effect on tumor growth. As expected, trastuzumab showed a partial inhibitory effect on tumor growth in the mouse group treated with clodrosome, when compared with the mice treated with clodrosome and isotype IgG. These results suggest that in addition to macrophages, other mechanisms of action were also involved in the tumor inhibition efficacy of trastuzumab. Collectively, these results suggest that trastuzumab treatment stimulated the overall immune response in mice as reflected by the increased macrophage population in the spleen. More importantly, infiltration of macrophages in tumor sites in mouse xenograft tumors was strongly suggested to be important for the efficacy of trastuzumab. To support this finding, splenocytes were collected from the four treatment groups and an ex vivo assay of cancer cell killing was conducted in the presence of trastuzumab. The results showed that splenocytes from trastuzumab-treated mice exhibited stronger cancer cell killing activity in the presence of trastuzumab than did that of the splenocytes from isotype IgG-treated mice (Fig. 3C). Splenocytes from the clodrosome-treated group exhibited significantly lower cancer cell killing activity in the presence of trastuzumab, suggesting the important role of macrophages in antitumor efficacy of trastuzumab in vivo.

FIGURE 3.
FIGURE 3.

The antitumor efficacy of trastuzumab depends on macrophage recruitment in the tumor tissues. BALB/c nu/nu mice (n = 5) were inoculated s.c. with 5 × 106 high HER2–expressing BT474 breast cancer cells. When tumors reached an average size of 100 mm3, mice were treated with the trastuzumab or an isotype control IgG weekly for a total of five doses. To deplete macrophages, tumor-bearing mice were injected i.p. with clodrosome 1 d before trastuzumab treatment for a total of 4 weekly dosing. (A) Macrophages (CD11b+) in the splenocytes and tumor-infiltrating cells of mice treated with trastuzumab or isotype control IgG with or without clodrosome treatment. A representative flow cytometry dot plot is shown for each condition. Percentages of macrophage are calculated and shown as mean ± SD. *p < 0.05. (B) Xenograft tumor sizes of the different treatment groups were measured twice weekly. Error bars indicate the SDs. n = 5. *p < 0.05, **p < 0.01. (C) The splenocytes were collected from each group and the cancer cell killing experiments were performed as described in Fig. 1 with an E:T ratio of 20:1. Cancer cell lysis mediated by the splenocytes in the presence of trastuzumab was calculated as described in Fig. 1. Error bars indicate the SD calculated from each group of mice. n = 3. *p < 0.05, **p < 0.01.

The FcγRIV plays a key role in macrophage-mediated ADCP and killing of Ab-opsonized cancer cells

Multiple FcγRs (FcγRI, II, III, and IV) are expressed on murine macrophages (21). With the exception of FcγRII, three of the four FcγRs are activating receptors for immune effector functions such as ADCP upon engagement of macrophage with the Ab Fc. To determine which FcγRs played a dominant role in macrophage-mediated ADCP and killing of Ab-opsonized cancer cells, we first measured the in vitro binding affinities of trastuzumab to the four mouse FcγRs by ELISA. As shown in Fig. 4A, trastuzumab showed concentration-dependent binding to all four mouse FcγRs, and trastuzumab binding to FcγRIV was the highest among the four murine FcγRs with an EC50 at 9.8 μg/ml. FcγRIV expression was then assessed in tumor-infiltrated immune cells by immunofluorescence staining. As shown in Fig. 4B, trastuzumab-treated tumor tissues showed higher FcγRIV expression than did that in IgG isotype-treated tumor tissues (Fig. 4B), indicating that more FcγRIV-expressing immune cells are recruited to the tumor sites. Flow cytometry analysis showed that M1 macrophages expressed the highest level of FcγRIV (mean fluorescence intensity [MFI] = 323), which was approximately twice the level of FcγRIV expression on BMMs (MFI = 165), M2 macrophages (MFI = 144), and Mcm macrophages (MFI = 192) (Fig. 4C). The high level of FcγRIV expression on M1 macrophages was consistent with the finding that M1 macrophages exhibited more potent killing of high HER2 cancer cells in the presence of trastuzumab than did BMMs, M2 macrophages, and Mcm macrophages (Fig. 2C).

FIGURE 4.
FIGURE 4.

FcγRIV expression is associated with trastuzumab anticancer efficacy. (A) FcγRIV shows stronger binding affinity to trastuzumab in comparison with other mouse FcγRs. The x-axis is at log scale and data points are average of three replicates by ELISA. Error bars indicate the SDs. (B) Higher FcγRIV expression was detected in tumor-infiltrated immune cells treated with trastuzumab than that of the isotype control by immunofluorescence. OCT frozen tumor tissues were stained with a rabbit anti-FcγRIV Ab and detected with an Alexa Fluor 488–anti-rabbit IgG Ab. A representative image is shown. Original magnification ×40. (C) FcγRIV expression on different types of macrophages by flow cytometry using the same Ab set as in (B). MFI is shown in the histogram.

To determine the function of FcγRIV expression on macrophages in trastuzumab-mediated ADCP activity, FcγRIV expression on the murine macrophage RAW264.7 cells was either knocked down (KD) by the shRNA lentivector system or upregulated by the addition of IFN-γ. As determined by flow cytometry, FcγRIV expression in the FcγRIV KD RAW264.7 cells was reduced to minimum levels (MFI = 4) in comparison with that in the wild-type (WT) cells (MFI = 25), whereas FcγRIV expression in response to IFN-γ stimulation was significantly upregulated (MFI = 75) (Fig. 5A). Cancer cell killing by FcγRIV KD RAW264.7 cells with was significantly reduced (16%) when compared with that by the WT RAW 264.7 cells (24%) (Fig. 5B). In contrast, increased FcγRIV expression resulting from IFN-γ stimulation showed stronger cancer cell killing (43%) in the presence of trastuzumab (Fig. 5B). We also compared the ADCP activity between FcγRIV KD, IFN-γ–stimulated, and WT RAW264.7 cells. The results showed that FcγRIV KD cells had reduced ADCP activity in comparison with WT RAW264.7 cells, whereas increased FcγRIV expression by IFN-γ stimulation resulted in stronger ADCP mediated by trastuzumab (Fig. 5C). Similar to the results from the RAW264.7 cells, BMMs showed increased FcγRIV by flow analysis when treated with IFN-γ (Fig. 5D), and IFN-γ–stimulated BMMs had stronger killing of high HER2 cancer cells and ADCP activity than did that of the control BMMs in the presence of trastuzumab (Fig. 5E, 5F). Collectively, these results suggest that FcγRIV expression level plays a key role in macrophage-mediated, trastuzumab-dependent killing of cancer cells.

FIGURE 5.
FIGURE 5.

FcγRIV expression on macrophage cells is associated with ADCP and cancer killing mediated by trastuzumab. (A) FcγRIV expression was detected by flow cytometry in WT, FcγRIV KD, and WT plus IFN-γ macrophage cells. MFI as shown in respective histograms. (B) Cancer cell killing by WT, FcγRIV KD, and WT plus IFN-γ macrophage cells in the presence of trastuzumab. The percentage of cell killing is calculated using the cell index collected by the xCELLigence instrument after 24 h of trastuzumab treatment. (C) ADCP activity of WT, FcγRIV KD, and WT plus IFN-γ macrophage cells in the presence of trastuzumab. The percentage of phagocytosis was calculated from the double-positive cells by flow cytometry as described in Fig. 2A. The results are shown as mean ± SD; n = 3. *p < 0.05. (D) FcγR IV expression in BMMs with or without IFN-γ (50 ng/ml) stimulation for 24 h by flow cytometry. (E) Cancer cell lysis mediated by BMMs or IFN-γ–stimulated BMMs in the presence of trastuzumab. (F) ADCP activity of BMMs and IFN-γ–stimulated BMMs mediated by trastuzumab as determined by flow cytometry. Percentage of phagocytosis was calculated as described in Fig. 2A. All experiments were repeated three times and error bars in the graphs show the SD. *p < 0.05.

Effect of human FcγRIII, the mouse FcγRIV ortholog, on the phagocytic activity of human macrophages

Human FcγRIII (CD16) shares 60% similarity in amino acid sequence with mouse FcγRIV/CD16-2 (22, 23). To investigate whether FcγRIII expressed on human macrophages have a similar effect on trastuzumab-mediated antitumor efficacy, we compared binding affinity (Kd) of trastuzumab to human FcγRIII and murine FcγRIV by the surface plasmon resonance method. The Kd to the murine FcγRIV (1.6 × 10−8 M) was >15-fold higher than that to the human FcγRIII (2.7 × 10−7 M). To determine the functional similarity between human FcγRIII and murine FcγRIV, we treated human monocyte cells (THP-1 cells) with human IFN-γ and determined FcγRIII expression by flow cytometry. Similar to the increased expression of FcγRIV in response to IFN-γ stimulation of mouse macrophages, FcγRIII expression on human THP-1 monocyte cells was also increased after treatment with IFN-γ (MFI = 307) when compared with the nonstimulated control THP-1 cells (MFI = 191) (Fig. 6A). ADCP activity of THP-1 monocyte cells in the presence of trastuzumab also increased when cells were stimulated with IFN-γ (Fig. 6B), suggesting that the increased FcγRIII expression in human macrophage cells can promote the trastuzumab-triggered phagocytosis of cancer cells.

FIGURE 6.
FIGURE 6.

Upregulation of human FcγRIII expression by IFN-γ stimulation in human monocytes increases ADCP activity in the presence of trastuzumab. (A) FcγRIII expression in THP-1 cells with or without IFN-γ stimulation was analyzed by flow cytometry. MFI is shown in the histogram. (B) Phagocytosis activity by THP-1 cells treated with or without IFN-γ in the presence of trastuzumab or isotype IgG. The results are shown as mean ± SD. n = 3. *p < 0.05.

Discussion

Immunotherapeutic Abs targeting surface Ags on tumor cells can simultaneously recruit immune effector cells to specifically destroy the malignant cells, an outcome that is mediated by interactions of the IgG Fc region with FcγRs on immune cells. Studies have shown that higher affinity interactions between Ab and FcγRs promote more robust ADCC and ADCP activities and are associated with better therapeutic efficacy of the anti-CD20 Ab rituximab (24, 25). Although it had been established that ADCC, mediated by NK cells, contributes to the efficacy of trastuzumab (26–28), a contribution of macrophage-mediated ADCP to antitumor efficacy of trastuzumab in vivo has not been reported. In this study, we demonstrated that activated macrophages readily phagocytosed trastuzumab-coated cancer cells (ADCP) and mediated Ab-dependent cancer cell killing in coculture conditions in vitro, and the results are consistent with the study reported previously (16). More importantly, our results demonstrated that monocytes/macrophages contributed to the efficacy of trastuzumab against HER2-expressing tumors by ADCP in vivo. In the high HER2–expressing BT474 mouse xenograft tumor model, the infiltration of macrophages into the tumor was increased in response to the trastuzumab treatment, suggesting that the trastuzumab Ab can recruit and engage macrophages at the tumor site. We also showed that a depletion of macrophages by clodrosome significantly impaired the in vivo antitumor efficacy of trastuzumab. Collectively, these results indicate that macrophages contribute to the antitumor efficacy of trastuzumab both in vitro and in vivo.

The depleting macrophages partially impaired antitumor efficacy of trastuzumab, and the data suggested that other mechanisms of action were also involved in the tumor-inhibiting efficacy of trastuzumab such as inhibition of HER2 signaling. In the nude mouse tumor model, NK cells are functional and can serve as effector cells in response to trastuzumab. Therefore, antitumor efficacy of trastuzumab in the mouse group with macrophage depletion by clodrosome might be contributed by an NK cell–mediated ADCC effect, as ADCC as a mechanism of action for trastuzumab was reported previously (27, 29). Other mechanisms of action of trastuzumab, including downregulation of HER2 expression (6) and inhibition of HER2 oncogenic signaling (2, 4), might also be involved.

IgG Abs interact with a family of FcγRs on immune cells and trigger immune effector functions such as ADCC and ADCP against infectious or pathological cells such as cancer cells (30, 31). Studies have shown that mice with Fcγ-chain knockout (γ−/−) or knockout of individual activating FcγRs lost immune effector functions of Abs and resulted in reduced antitumor efficacy of trastuzumab (22, 32, 33). Mouse effector cells express four different FcγRs: FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV (CD16-2). FcγRIV is a newer member of the family and maps in the 75-kb genomic interval between FcγRII and FcγRIII; its expression is restricted to myeloid lineage cells (34). Studies have demonstrated that different IgG subclasses selectively interact with certain activating FcγRs in vitro and in vivo (35). FcγRIV binds to mouse IgG2a and IgG2b with intermediate affinity, but it has no detectable binding to mouse IgG1 or IgG3 (22). It has been shown that FcγRIV is important in mediating the function of anticancer Ab (IgG2a) in the model of lung and liver metastases using murine melanoma cells, and in an anti-CD20 Ab-dependent B cell depletion model (24, 36, 37). Interestingly, we found that human IgG1 exhibited the strongest binding to murine FcγRIV in comparison with the binding affinity to the other murine FcγRs, including the generally considered high-affinity FcγRI. This finding has significant implications for our understanding of immune cell engagement by therapeutic Abs of the human IgG1 isotype in mouse xenograft tumor models, because human or humanized Abs in preclinical studies are often evaluated in mouse xenograft tumor models where mouse FcγRs interact with the human IgG1 such as trastuzumab. We showed that FcγRIV-expressing immune cells in tumor tissue were increased in response to trastuzumab treatment in the high HER2 breast cancer xenograft tumor model and demonstrated that FcγRIV expression on the macrophage is associated with ADCP and cancer cell killing. Knockdown of FcγRIV in macrophages significantly reduced trastuzumab-mediated ADCP and cancer cell killing, whereas increased FcγRIV expression on macrophages by IFN-γ stimulation enhanced ADCP and killing of cancer cells in the presence of trastuzumab. Taken together, these results demonstrated that FcγRIV played an important role in immune cell engagement by human IgG1 Abs such as trastuzumab.

Human immune cells have a corresponding family of FcγRs, including FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), and FcγRIII (CD16A/B) (35). On the basis of protein sequence and function, human FcγRIII is often considered the ortholog of mouse FcγRIV (22, 23). Similar to the function of mouse FcγRIV, we showed that FcγRIII expression on human monocytes and macrophages was also increased in response to IFN-γ stimulation, and also resulted in increased ADCP activity and cancer cell killing triggered by trastuzumab. It is well established that immune effector functions depend on the balance of both activating and inhibiting FcγRs. These results indicate that FcγRIII may play an important role in human macrophage-mediated ADCP activity. Clinical studies have implicated the important role of FcγRIII in trastuzumab efficacy (38), even though function of FcγRIII on NK cells is considered mainly to contribute to cancer cell killing through ADCC (39). Future studies are warranted to establish the roles of ADCP mediated by macrophages in trastuzumab anticancer efficacy in the clinic. Taken together, this study demonstrated that trastuzumab can trigger ADCP and that increased expression of mouse FcγRIV/human FcγRIII promotes the ADCP activity of macrophages triggered by the Ab. Our results suggest that modulation of macrophages by stimulation of the activating FcγRIII levels can serve as a strategy for enhancing Ab-mediated ADCP and ADCC for improved efficacy of anticancer Ab immunotherapies.

Disclosures

R.J.B., M.R., R.E.J., and W.R.S. were employees of Janssen Research & Development, LLC at the time the studies were undertaken. The remaining authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Qingchun Tong for providing mouse tissue for bone marrow cell isolation. We also thank Dr. Amy Lauren Hazen for expert assistance in flow analysis.

Footnotes

  • This work was supported by grants from Janssen Research & Development, LLC and the Texas Emerging Technology Fund, Welch Foundation Grant AU0042, and by a flow cytometry user support award from the Cancer Prevention and Research Institute of Texas (Grant RP110776).

  • Abbreviations used in this article:

    ADCC
    Ab-dependent cell-mediated cytotoxicity
    ADCP
    Ab-dependent cellular phagocytosis
    BMM
    bone marrow–derived macrophage
    KD
    knocked down
    M1
    type 1 macrophage
    M2
    type 2 macrophage
    Mcm
    macrophage derived with cancer cell–conditioned medium
    MFI
    mean fluorescence intensity
    shRNA
    short hairpin RNA
    TAM
    tumor-associated macrophage
    WT
    wild-type.

  • Received November 17, 2014.
  • Accepted February 19, 2015.

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