IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production

Abstract

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.

FIGURE 1

IL-12 enhances the effects of an anti-HER2 mAb in a murine tumor model. A, Mice with s.c. CT-26^HER2/neu tumors were treated i.p. with PBS, 1 μg IL-12, 1 mg/kg 4D5 (a murine mAb recognizing human HER2), or IL-12 plus 4D5 (same doses). Tumor volumes were calculated as described in Materials and Methods. Standard error was <5% for each data point shown. This experiment was repeated five times with similar results. B–D, Serum was harvested from each of the mice 24 h after the final administration of IL-12 and 4D5, and an ELISA technique was used to measure levels of the following cytokines: (B) IFN-γ, (C) RANTES, and (D) IL-8. *p <0.01 versus all conditions shown.

FIGURE 2

Antitumor effects of IL-12 and 4D5 are dependent on IFN-γ. Wild-type or IFN-γ–deficient mice (IFN-γ^−/−) bearing CT-26^HER2/neu tumors were treated with PBS or the combination of IL-12 (1 μg) and 4D5 (1 mg/kg). Tumor volumes were calculated as described in Materials and Methods. Standard error was <5% for each data point shown. This experiment was repeated twice with similar results.

FIGURE 3

Antitumor effects of IL-12 and 4D5 are dependent on NK cells. A, PBS or the combination of IL-12 and 4D5 was administered to CT-26^HER2/neu tumor-bearing mice that had been depleted of NK cells via administration of anti-asialo GM1 (see Materials and Methods for depletion protocol). Mock-treated mice received an isotype-matched control Ab. This treatment resulted in >95% depletion of NK cells from the peripheral blood and spleen, as determined by flow cytometry (data not shown). B, Serum was harvested from each of the mice 24 h after the final dose of IL-12 and 4D5 and analyzed for IFN-γ content by ELISA. *p <0.001 versus all conditions shown. This experiment was repeated twice with similar results.

FIGURE 4

IFN-γ does not inhibit proliferation of CT-26^HER2/neu cells. A, CT-26^HER2/neu cells were treated in vitro with IL-12, trastuzumab, or increasing concentrations of IFN-γ. Proliferation was determined by the MTT assay. B, CT-26^HER2/neu cells were stimulated for 15 min in vitro with increasing concentrations of IFN-γ or PBS. The percentage of cells containing activated STAT1 was assessed by flow cytometry. The x-axis of each histogram represents the specific fluorescence of p-STAT1 on a four-decade logarithmic scale, and the y-axis represents the total number of events. C, CT-26^HER2/neu cells and parental CT-26 cells were analyzed for the binding of 4D5 to cell surface HER2/neu by flow cytometry using the 4D5 Ab and an FITC-labeled rabbit anti-murine Ab.

FIGURE 5

Lymphocyte infiltration of tumors is similar in the four treatment groups. CT-26^HER2/neu tumors from mice treated with PBS, IL-12, 4D5, or a combination of both agents were stained for (A) CD4⁺ lymphocytes and (B) CD8⁺ lymphocytes. Representative tumor sections from each treatment group are shown with arrows depicting Novared positive CD4 and CD8 stained T cells. Original magnification ×40 (all fields).

FIGURE 6

Histopathology and electron microscopy analysis of tumor tissues. Tumor sections obtained on day 21 of the study were analyzed for necrosis by H&E staining and electron microscopy as described in Materials and Methods. A, A representative H&E-stained tumor section from mice treated with PBS, IL-12, 4D5, or IL-12 plus 4D5 is shown with arrows indicating regions of necrosis. Original magnification ×40 (all fields). B, Electron micrographs of tumors treated with PBS, IL-12, 4D5, or IL-12 plus 4D5. PBS-treated tumors contain dividing tumor cells surrounded by a clear extracellular space (asterisk). 4D5-treated tumors had similar characteristics. Tumor cells from IL-12–treated mice had nuclei with dispersed heterochromatin (X) and membrane containing autophagocytic vesicles (arrows). Tumors from mice receiving dual therapy exhibited high levels of extracellular debris, swollen mitochondria, and fragments of dead cells. Scale bar, 1 μm. C, Photomicrographs of H&E-stained day 15 tumor sections from wild-type and IFN-γ–deficient mice treated with IL-12 plus 4D5. Xenografts in treated knockout mice were smaller and exhibited minimal coagulation necrosis (asterisks).

FIGURE 7

Analysis of NK cell activation status. A, Immunofluorescent microscopy for CD335 (NKp46, an NK cell activation marker, in red) in tumors of treated mice. Tumor sections counterstained with DAPI for nuclear identification. Original magnification ×40. B, Flow cytometry for CD49b and CD69 in the splenocytes of treated mice.

FIGURE 8

Expression of IFN-γ–induced anti-angiogenic factors. A, Circulating levels of MIG were measured by ELISA in the plasma of treated mice on day 13. B, Overnight culture of 2 × 10⁵ CT-26^HER2/neu tumor cells with IFN-γ (1 or 10 ng/ml) led to increased production of IP-10 in supernatants as measured by ELISA. C, Forty-eight hour coculture of tumor cells (5 × 10⁴ cells) with day 13 splenocytes (2 × 10⁵ cells) isolated from combination-treated mice led to increased production of IP-10 compared with that using splenocytes from control-treated mice.