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. 2018 Dec 31;13(12):e0200664.
doi: 10.1371/journal.pone.0200664. eCollection 2018.

Anti-CD137 monoclonal antibody enhances trastuzumab-induced, natural killer cell-mediated cytotoxicity against pancreatic cancer cell lines with low human epidermal growth factor-like receptor 2 expression

Takushi Masu  1 , Masanori Atsukawa  1 , Katsuhisa Nakatsuka  1 , Masumi Shimizu  2 , Daishu Miura  3 , Taeang Arai  1 , Hirotomo Harimoto  1 , Chisa Kondo  1 , Keiko Kaneko  1 , Seiji Futagami  1 , Chiaki Kawamoto  1 , Hidemi Takahashi  2 , Katsuhiko Iwakiri  1
Affiliations

Anti-CD137 monoclonal antibody enhances trastuzumab-induced, natural killer cell-mediated cytotoxicity against pancreatic cancer cell lines with low human epidermal growth factor-like receptor 2 expression

Takushi Masu et al. PLoS One. .

Abstract

Because human epidermal growth factor-like receptor (HER) 2 is expressed on the surface of human pancreatic carcinoma cells to varying degrees, trastuzumab, an anti-HER2 monoclonal antibody (mAb), is expected to exert antibody-dependent, natural killer (NK) cell-mediated cytotoxicity (ADCC) against the cells. However, some reports found that the effect of trastuzumab against human pancreatic carcinoma cells was limited because most express only limited HER2. We examined whether anti-CD137 stimulating mAb could enhance trastuzumab-mediated ADCC against Panc-1, a human pancreatic cancer cell line with low HER2 expression, in vitro. Supplementation of anti-CD137 mAb could improve trastuzumab-mediated ADCC against Panc-1 which was insufficient without this stimulating antibody. The ADCC differed in individual cells, and this was related to the expression of CD137 on the surface of NK cells after trastuzumab stimulation in association with the Fcγ-RIIIA polymorphism. NK cells with Fcγ-RIIIA-VV/VF showed high levels of ADCC against Panc-1, but those with Fcγ-RIIIA-FF did not show optimal ADCC. In addition, trastuzumab-mediated ADCC against the human pancreatic cancer cell line Capan-1 with high HER2 expression was generally high and not affected by the Fcγ-RIIIA polymorphism. These results demonstrated that in Fcγ-RIIIA-VV/VF-carrying healthy individuals, trastuzumab plus αCD137 mAb could induce effective ADCC against HER2-low-expressing pancreatic cancer cell lines, and that such an approach may result in similar findings in patients with pancreatic cancer.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
A: Flow cytometric analysis was performed to confirm HER2 expression on Panc-1 (upper panel) and Capan-1 (lower panel). B: The isolated NKs cell were confirmed to be CD3 negative and CD56 positive by flow cytometry. C: The fundamental viability of the isolated NK cells was evaluated as cytotoxicity against the NK-sensitive thymoma cell line K562.
Fig 2
Fig 2. LDH-releasing assays were performed to confirm the appropriate conditions for Tmab-mediated ADCC against Capan-1, a HER2-high-expressing human pancreatic cancer cell line.
A: The contribution of Tmab to ADCC against Capan-1 was confirmed. The effector/target (E/T) ratio was set at 10:1, and 10 μg/ml of Tmab and isotype IgG were used. B: To confirm the appropriate concentration of Tmab, ADCC with the indicated dose of Tmab was measured. The E/T ratio was set at 40:1. C: To determine the necessity for the preadministration of Tmab to target cells, Capan-1 was incubated with 10 μg/ml of Tmab for the indicated times. Cells were harvested and ADCC was evaluated. The E/T ratio was set at 40:1.
Fig 3
Fig 3. Tmab-mediated ADCC against Panc-1, a HER2-low-expressing human pancreatic cancer cell line, was investigated.
A: Mean ADCC against Panc-1 was compared with that against Capan-1. B: ADCC against Panc-1 and Capan-1 in a representative individual was shown.
Fig 4
Fig 4. To establish how to increase ADCC against a HER2-low-expressing pancreatic cancer cell line, the addition of mAbs with Tmab was investigated.
A: To select the optimal antibody, flow cytometric analysis was performed to evaluate changes in PD-1, NKG2D and CD137 on the surface of NK cells after Tmab administration. Based on the results that only CD137 was up-regulated after Tmab adoministration, αCD137 was used in the following examinations. B: The effect of αCD137 combined with Tmab on ADCC against Panc-1 was evaluated. Mean ADCC in the indicated E/T ratios is shown. C: Results in a representative individual cell when ADCC increased with the addition of αCD137. Left two histograms: Changes in the expression of CD137 on the surface of NK cells. Right panel: Increase in Tmab-mediated ADCC with the addition of αCD137. D: Results in an unresponsive individual cell.
Fig 5
Fig 5. Effects of FcγRIIIA polymorphisms on the activity of NK cells.
FcγRIIIA polymorphisms were determined by flow cytometric analysis and divided into the VV/VF (n = 8) and FF groups (n = 4). Differences in CD137 expression on the surface of NK cells in both groups were measured with flow cytometry at the end of Tmab-induced ADCC against Panc-1 (5A, left panel) and Capan-1 (5A, right panel). Levels of IFN-γ (5B, upper panels) and TNF-α (5B, lower panels) released from NK cells at the end of the ADCC assay were measured using ELISA.
Fig 6
Fig 6. Effects of αCD137 addition to Tmab on the ADCC against Panc-1 (6A, left panel) and Capan-1 (6A, right panel) were compared between VV/VF and FF individuals.
The percentage of increase in ADCC after the addition of anti-CD137 mAb against Panc-1 (6B, left panel) and Capan-1 (6B, right panel) in each individual were calculated as indicated.
Fig 7
Fig 7. To determine the contribution of Tmab and αCD137 mAb for degranulation from NK cells, changes of intracellular perforin and granzyme-B were analyzed when NK cells obtained from FcγRIIIA-VV carrying healthy individuals were incubated with HER2-high-expressing human pancreatic cancer cell line, Capan-1 in the presence of Tmab and/or aCD137 mAb.
The upper 5 panels show the percentage of perforin and granzyme-B positive cells in each culture conditions. The activation of NK cells in each condition was simultaneously evaluated by the expression of CD69 on the surface of NK cells and shown in lower 5 histograms. The results shown in this figure were obtained from the representative individual.
Fig 8
Fig 8. Schema illustrated the contribution of aCD137 and FcγR-III polymorphism for trastuzumab-mediated ADCC against HER2-low-expressing human pancreas cancer cell line.
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