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. 2018 Oct 15;13(10):e0204880.
doi: 10.1371/journal.pone.0204880. eCollection 2018.

Stimulation of natural killer cells with rhCD137 ligand enhances tumor-targeting antibody efficacy in gastric cancer

Toshihiro Misumi  1 , Kazuaki Tanabe  1 , Nobuaki Fujikuni  1 , Hideki Ohdan  1
Affiliations

Stimulation of natural killer cells with rhCD137 ligand enhances tumor-targeting antibody efficacy in gastric cancer

Toshihiro Misumi et al. PLoS One. .

Abstract

Although many anticancer agents for gastric cancer have been developed, the prognosis for many patients remains poor. Recently, costimulatory immune molecules that reactivate antitumor immune responses by utilizing the host immune system have attracted attention as new therapeutic strategies. CD137 is a costimulatory molecule that reportedly potentiates the antitumor activity of tumor-targeting monoclonal antibodies (mAbs) by enhancing antibody-dependent cellular cytotoxicity. However, it remains unclear whether CD137 stimulates tumor-regulatory activity in gastric cancer. In this study, we investigated the antitumor effects of CD137 stimulation on gastric cancer cells administered tumor-targeting mAbs. Our results showed that human natural killer (NK) cells were activated by expressing CD137 after encountering trastuzumab-coated gastric cancer cells, and that stimulation of activated NK cells in the presence of trastuzumab and recombinant human CD137 ligand (rhCD137L) enhanced cytotoxicity and release of cytokines (IFN-γ, TNF, granzyme A, or granzyme B) as compared with activated NK cells with trastuzumab alone (p < 0.05). By combination treatment with rhCD137L, similar effects were obtained regarding cancer cell cytotoxicity in the presence of cetuximab (p < 0.01). Moreover, we revealed that CD137 expression was dependent upon the affinity between the Fc portion of the antibodies and FcγRIIIa of NK cells based on results indicating that human IgG1 and IgG3 subclasses enhanced CD137 expression (p < 0.001). These results confirmed that FcγRIIIA polymorphisms (158 V/V) enhanced CD137 expression to a greater degree than 158 F polymorphisms (p = 0.014). Our results suggested that CD137 stimulation could promote the effects of tumor-targeting mAbs in gastric cancer, and that further investigation of antibody binding affinity and in vivo activities might improve therapeutic strategies related to the treatment of gastric cancer patients.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. CD137 expression on NK cells following incubation of gastric cancer cells with trastuzumab.
Purified NK cells from PBMCs of healthy individuals were analyzed for CD137 expression after 24-h culture with gastric cancer cell lines and trastuzumab. (A) HER2 expression on gastric cancer cell lines (TMK-1, NCI-N87, GLM-5S, and GLM-4). (B) CD137 expression on CD3-CD56+ NK cells from a representative healthy individual after 24-h culture with the respective gastric cancer cell lines in the presence or absence of trastuzumab. (C) Mean fluorescence intensity (MFI) of CD137 expression on NK cells from five healthy individuals cultured with the respective gastric cancer cell lines, * p < 0.001. Data are shown as the mean ± SEM.
Fig 2
Fig 2. Enhancement of trastuzumab-mediated NK cell cytotoxicity by rhCD137L.
To evaluate NK cell cytotoxic function, purified NK cells from three healthy individuals were incubated with chromium-labeled tumor cells, including (A) GLM-4, (B) NCI-N87, and (C) TMK-1 for 18 h with medium alone, trastuzumab (10 ng/mL), or trastuzumab plus rhCD137L (250 ng/mL). The lysis percentages of the target cells cultured with medium alone (circles), trastuzumab (squares), or trastuzumab plus rhCD137L (triangles) according to chromium release at varying effector (NK cells):target-cell ratios are shown. (A, *p = 0.03; B, *p < 0.01; C, p = not significant). Data are shown as the mean ± SEM.
Fig 3
Fig 3. Increased cytokine secretion of trastuzumab-treated NK cells by rhCD137L administration.
To investigate NK cell degranulation and cytokine secretion, NK cells were incubated with GLM-4 in the following four conditions: medium alone, trastuzumab (10 ng/mL), rhCD137L (250 ng/mL), or trastuzumab (10 ng/mL) with rhCD137L (250 ng/mL). (A) A representative flow cytometric plot of CD56 and CD107a double staining. Percentage and MFI of CD107a-expressing NK cells from five healthy individuals [p = not significant (NS)]. (B) Cytokine secretion (human IFN-γ, TNF, granzyme A, or granzyme B) determined by the cytometric bead array (*p < 0.005). Data are shown as the mean ± SEM.
Fig 4
Fig 4. Enhancement of cetuximab-mediated NK cell cytotoxicity by rhCD137L administration.
Purified NK cells were analyzed for cytotoxicity with cetuximab (10 ng/mL) plus rhCD137L (250 ng/mL) in a chromium-release assay, as described previously. NK cells were incubated with gastric cell lines expressing high levels of EGFR [(A) MKN-1 and (B) MKN-45] or low levels of EGFR [(C) TMK-1] under the same conditions. Lysis percentages of the target cells cultured with medium alone (circles), cetuximab (squares), or cetuximab and rhCD137L (triangles) according to chromium release at varying effector (NK cells)/target-cell ratios are shown. (A, B, *p < 0.01; C, p = not significant). Data are shown as the mean ± SEM.
Fig 5
Fig 5. Upregulated CD137 expression and cytotoxicity of NK cells by treatment with a combination of trastuzumab and pertuzumab.
(A) To evaluate upregulated CD137 expression in NK cells by treatment with the combination of dual anti-HER2 mAbs that bind different domains, purified NK cells were incubated with a HER2-expressing gastric cancer cell line (GLM-4) in medium containing various trastuzumab and pertuzumab concentrations (0–10 μg/mL) for 24 h. The MFI of CD137-expressing NK cells from three healthy individuals was analyzed by flow cytometry (*p < 0.05). Data are shown as the mean ± SEM. (B) Purified NK cells were analyzed for cytotoxicity in a chromium-release assay. Chromium-labeled GLM-4 cells and NK cells cultured with medium alone (circles), pertuzumab (white diamonds), trastuzumab (black diamonds), pertuzumab and trastuzumab (squares), or dual anti-HER2 mAbs and rhCD137L (triangles) are shown. (*p < 0.01). Data are shown as the mean ± SEM.
Fig 6
Fig 6. Upregulated CD137 expression induced by incubation with immobilized IgG subclass.
Purified NK cells were incubated for 24 h in the presence of immobilized IgG subclass (2 mg/mL), and CD137 expression was measured by flow cytometry. (A) CD137 expression on NK cells from a representative healthy individual after a 24-h culture. Percentages of CD137-expressing NK cells per quadrant are indicated. (B) MFI of CD137-expressing NK cells from five healthy individuals. **p < 0.001; *p = 0.016. Data are shown as the mean ± SEM.
Fig 7
Fig 7. The influence of FcγRIIIa polymorphisms on trastuzumab-induced CD137 expression.
Percentage of CD137-expressing NK cells from 38 healthy individuals after a 24-h incubation with trastuzumab-coated gastric cancer cells (GLM-4) exhibiting FcγRIIIa 158 polymorphism. CD137 presentation on NK cells was compared between V/V (n = 19) and F carriers (n = 19). F carriers were also divided into two groups: VF (n = 15, circles) and FF (n = 4, diamonds). *p = 0.014. Data are shown as the mean ± SEM.
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The Japan Society for the Promotion of Science (JSPS) KAKENHI (grant No. 25462023) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.