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. 2020 Aug;8(2):e000873.
doi: 10.1136/jitc-2020-000873.

Anti-tumor effects of NK cells and anti-PD-L1 antibody with antibody-dependent cellular cytotoxicity in PD-L1-positive cancer cell lines

Ji-Eun Park  1 , Seong-Eun Kim  2 , Bhumsuk Keam  3   4 , Ha-Ram Park  1 , Soyeon Kim  1   5 , Miso Kim  1   4 , Tae Min Kim  1   4 , Junsang Doh  6 , Dong-Wan Kim  1   4 , Dae Seog Heo  1   4
Affiliations

Anti-tumor effects of NK cells and anti-PD-L1 antibody with antibody-dependent cellular cytotoxicity in PD-L1-positive cancer cell lines

Ji-Eun Park et al. J Immunother Cancer. 2020 Aug.

Abstract

Background: Although programmed cell death-1/programmed death-ligand 1 (PD-L1) inhibitors show remarkable antitumor activity, a large portion of patients with cancer, even those with high PD-L1-expressing tumors, do not respond to their effects. Most PD-L1 inhibitors contain modified fragment crystallizable region (Fc) receptor binding sites to prevent antibody-dependent cellular cytotoxicity (ADCC) against PD-L1-expressing non-tumor cells. However, natural killer (NK) cells have specific antitumor activity in the presence of tumor-targeting antibody through ADCC, which could enhance NK cell-induced cytotoxicity. We evaluated the antitumor efficacy of ADCC via anti-PD-L1 monoclonal antibodies (mAbs) and NK cells against several PD-L1-positive cancer cell lines.

Methods: Various cancer cell lines were used as target cell lines. Surface PD-L1 expression was analyzed by flow cytometry. IMC-001 and anti-hPD-L1-hIgG1 were tested as anti-PD-L1 mAbs with ADCC and atezolizumab as an anti-PD-L1 mAb without ADCC. NK cell cytotoxicity was measured by 51Cr-release assay and CD107a degranulation assay. Also, live cell imaging was performed to evaluate cytotoxicity in a single-cell level. NK-92-CD16 (CD16-transduced NK-92 cell line) and peripheral blood mononuclear cells from healthy donors, respectively, were used as an effector cell. FcγRIIIa (CD16a)-V158F genotyping was performed for healthy donors.

Results: We demonstrated that the cytotoxicity of NK-92-CD16 cells toward PD-L1-positive cancer cell lines was significantly enhanced in the presence of anti-PD-L1 mAb with ADCC. We also noted a significant increase in primary human NK cell cytotoxicity against PD-L1-positive human cancer cells when cocultured with anti-PD-L1 mAb with ADCC. Moreover, NK cells expressing a FCGR3A high-affinity genotype displayed higher anti-PD-L1 mAb-mediated ADCC lysis of tumor cells than donors with a low-affinity genotype.

Conclusion: These results suggest that NK cells induce an ADCC response in combination with anti-PD-L1 mAbs, which helps promote ADCC antitumor activity against PD-L1-positive tumors. This study provides support for NK cell immunotherapy against high PD-L1-expressing tumors in combination with ADCC through anti-PD-L1 mAbs.

Keywords: cytotoxicity; head and neck neoplasms; immunological; immunotherapy; killer cells; lung neoplasms; natural.

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Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1
Surface PD-L1 expression in various cancer cell lines. (A) PD-L1 mRNA expression in various cancer cell lines was determined by Affymetrix expression data in the Cancer Cell Line Encyclopedia database. Bars are colored by cancer type: DLBCL and RCC. (B) Expression levels of surface PD-L1 measured by flow cytometry. Bar graphs show ΔMFI averaged from three independent experiments. (C) Representative histogram showing isotype control (gray shaded) and staining with an anti-PD-L1 antibody (blue line). Data shown are mean±SD. ΔMFI, delta mean fluorescence intensity; DLBCL, diffuse large B-cell lymphoma; HNSCC, head and neck squamous cell carcinoma; NSCLC, non-small-cell lung cancer; PD-L1; programmed death-ligand 1; RCC, renal cell carcinoma.
Figure 2
Figure 2
Anti-PD-L1 mAbs-mediated ADCC in human cancer cell lines. NK-92-CD16 cytotoxicity against tumor cells was measured by a standard 51Cr-release assay with various E:T ratios (30:1, 10:1, and 3:1). Bar graph represents cytotoxicity of NK-92-CD16 cells against cancer cell lines at 30:1 E:T ratio. All cancer cell lines were treated with 10 µg/mL of IgG1 isotype control (black dotted lines and bar), atezolizumab (green), IMC-001 (red), and anti-hPD-L1-hIgG1 (blue). (A) Head and neck squamous cell carcinoma cell lines. PD-L1 low (left) includes SNU-1041, SNU-1066, and Detroit 562, and PD-L1 high (right) includes SNU-1076, FaDu, and HN31 cells. (B) For non-small-cell lung cancer, NCI-H1650 cells are PD-L1 low (left), and NCI-H1975 cells are PD-L1 high (right). All experiments were performed three times independently. Statistical significance across groups 6 was determined by one-way analysis of variance. All data are shown as mean±SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ADCC, antibody-dependent cellular cytotoxicity; E:T, effector-to-target; mAb, monoclonal antibody; NK, natural killer; ns, not significant. PD-L1; programmed death-ligand 1.
Figure 3
Figure 3
Enhanced cytotoxicity of NK cells against human cancer cells through anti-PD-L1 mAb-mediated ADCC. Cytotoxicity of primary NK cells mediated by anti-PD-L1 mAb ADCC was measured in human head and neck squamous cell carcinoma and non-small-cell lung cancer cell lines using a CD107a degranulation assay. PBMCs from healthy donors were used for effector cells both in resting (black) and activated (blue) states (n=8). PBMCs were cultured overnight or activated with 1 ng/mL interleukin-15 for 3 days. Cancer cell lines were pretreated with anti-PD-L1 mAbs for 30 min. After washing, human cancer cell lines were cocultured with effector cells 1 hour at ratio of 1:1. (A) Results of CD107a degranulation assay in PD-L1 low-expressing and (B) PD-L1 high-expressing cancer cell lines. Statistical significance across groups was determined by one-way analysis of variance. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ADCC, antibody-dependent cellular cytotoxicity; mAb, monoclonal antibody; NK, natural killer; ns, not significant; ns, not significant; PBMC, peripheral blood mononuclear cell; PD-L1; programmed death-ligand 1.
Figure 4
Figure 4
Effect of FCGR3A polymorphism on IMC-001-mediated antibody-dependent cellular cytotoxicity with NK cells. To compare the cytotoxicity of NK cells between high-affinity genotyped (V/V or V/F genotype) with low affinity genotyped (F/F genotype), we defined y-axis as ΔCD107a+ percentage of NK cells subtracting control from IMC-001 CD107a+ percentage of NK cells. Healthy donors were genotyped for FCGR3A-158 polymorphisms by PCR-based Sanger sequencing (n=6 or 8). Human cancer cell lines were used as targets with high-affinity (n=4; Detroit 562, HN31 and NCI-H1975 n=3) or low-affinity NK cell genotypes. Human (A) head and neck squamous cell carcinoma and (B) non-small-cell lung cancer cell lines classified by expression level of PD-L1 (PD-L1 high-expressing (right) and low-expressing (left) cancer cell lines). The status of primary NK cells are grouped as resting primary (black) and activated (blue) cells. A one-tailed unpaired Student’s t-test was used to compare statistical significance. *P<0.05, **P<0.01, ***P<0.001. NK, natural killer; ns, not significant; PBMC, peripheral blood mononuclear cell; PD-L1; programmed death-ligand 1.
Figure 5
Figure 5
Live cell imaging-based analysis of NK cell cytotoxicity. (A) Representative time-lapse images of interaction between NK-92-CD16 cells (yellow lines) and HN-31 cells (white lines) treated with various antibodies. (B–D) Effects of antibody treatment on overall cytotoxicity (B), time for killing (C), and number of cancer cells killed by an NK cell (D). Mann-Whitney test was used. *P<0.05,**P<0.01, ***P<0.001. NK, natural killer; ns, not significant.
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