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. 2018 Dec 6:9:2873.
doi: 10.3389/fimmu.2018.02873. eCollection 2018.

Expression of a Recombinant High Affinity IgG Fc Receptor by Engineered NK Cells as a Docking Platform for Therapeutic mAbs to Target Cancer Cells

Kristin M Snyder  1 , Robert Hullsiek  1 , Hemant K Mishra  1 , Daniel C Mendez  1 , Yunfang Li  1 , Allison Rogich  1 , Dan S Kaufman  2 , Jianming Wu  1 , Bruce Walcheck  1
Affiliations

Expression of a Recombinant High Affinity IgG Fc Receptor by Engineered NK Cells as a Docking Platform for Therapeutic mAbs to Target Cancer Cells

Kristin M Snyder et al. Front Immunol. .

Abstract

Anti-tumor mAbs are the most widely used and characterized cancer immunotherapy. Despite having a significant impact on some malignancies, most cancer patients respond poorly or develop resistance to this therapy. A known mechanism of action of these therapeutic mAbs is antibody-dependent cell-mediated cytotoxicity (ADCC), a key effector function of human NK cells. CD16A on human NK cells has an exclusive role in binding to tumor-bound IgG antibodies. Though CD16A is a potent activating receptor, it is also a low affinity IgG Fc receptor (FcγR) that undergoes a rapid downregulation in expression by a proteolytic process involving ADAM17 upon NK cell activation. These regulatory processes are likely to limit the efficacy of tumor-targeting therapeutic mAbs in the tumor environment. We sought to enhance NK cell binding to anti-tumor mAbs by engineering these cells with a recombinant FcγR consisting of the extracellular region of CD64, the highest affinity FcγR expressed by leukocytes, and the transmembrane and cytoplasmic regions of CD16A. This novel recombinant FcγR (CD64/16A) was expressed in the human NK cell line NK92 and in induced pluripotent stem cells from which primary NK cells were derived. CD64/16A lacked the ADAM17 cleavage region in CD16A and it was not rapidly downregulated in expression following NK cell activation during ADCC. CD64/16A on NK cells facilitated conjugation to antibody-treated tumor cells, ADCC, and cytokine production, demonstrating functional activity by its two components. Unlike NK cells expressing CD16A, CD64/16A captured soluble therapeutic mAbs and the modified NK cells mediated tumor cell killing. Hence, CD64/16A could potentially be used as a docking platform on engineered NK cells for therapeutic mAbs and IgG Fc chimeric proteins, allowing for switchable targeting elements and a novel cancer cellular therapy.

Keywords: ADCC; FcR; NK cell; antibody; immunotherapy.

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Figures

Figure 1
Figure 1
Expression of CD64/16A by NK92 cells. (A) Schematic representation of the cell membrane forms of CD16A, CD64, and CD64/16A. CD16A undergoes ectodomain shedding by ADAM17 at a membrane proximal location, as indicated, which is not present in CD64 and CD64/16A. (B) NK92 parental cells, NK92-CD16A cells, and NK92-CD64/16A cells were stained with an anti-CD16, anti-CD64, or an isotype-matched negative control mAb and examined by flow cytometry. (C) NK92-CD16A and NK92-CD64/16A cells were incubated with SKOV-3 cells with or without trastuzumab (5 μg/ml) at 37°C (E:T = 1:1; 5 × 105 cells to 5 × 105 cells) for 2 h. The NK92-CD16A and NK92-CD64/16A cells were then stained with an anti-CD16 mAb or an anti-CD64 mAb, respectively, and examined by flow cytometry. Non-specific antibody labeling was determined using the appropriate isotype-negative control mAb. (B,C) Data is representative of at least three independent experiments.
Figure 2
Figure 2
CD64/16A promotes target cell conjugation, ADCC, and IFNγ production. (A) NK92 control cells or NK92-CD64/16A cells, both expressing GFP, and SKOV-3 cells, labeled CellTrace Violet, were mixed at an E:T ratio of 1:2 (1 × 105 cells to 2 × 105 cells) in the presence or absence of trastuzumab (5 μg/ml) and the anti-CD64 mAb 10.1 (10 μg/ml), incubated at 37°C up to 60 min, fixed, and then analyzed by flow cytometry, as described in the Materials and Methods. For the flow cytometric data, representative data are shown. For the bar graphs, mean ± SD of three independent experiments is shown. Statistical significance is indicated as ***p < 0.001. (B) NK92-CD64/16A cells were incubated with SKOV-3 cells (E:T = 20:1; 1.6 × 105 cells to 8 × 103 cells) and trastuzumab (tras.) at the indicated concentrations (left panel), or with SKOV-3 cells at the indicated E:T ratios in the presence or absence of trastuzumab (5 μg/ml) (right panel) for 2 h at 37°C. Data are represented as % specific release and the mean ± SD of 3 independent experiments is shown. Statistical significance is indicated as *p < 0.05, **p < 0.01. (C) NK92-CD64/16A cells were incubated with SKOV-3 cells (E:T = 20:1; 1.6 × 105 cells to 8 × 103 cells) in the presence or absence of trastuzumab (5 μg/ml) and the anti-CD64 mAb 10.1 (10 μg/ml), as indicated, for 2 h at 37°C. Data are represented as % specific release and the mean ± SD of 3 independent experiments is shown. Statistical significance is indicated as **p < 0.01. (D) NK92-CD64/16A cells were incubated with SKOV-3 cells (E:T = 1:1; 1 × 105 cells to 1 × 105 cells) with or without trastuzumab (5 μg/ml) for 2 h at 37°C. Secreted IFNγ levels were quantified by ELISA. Data is shown as mean of 2 independent experiments.
Figure 3
Figure 3
CD64/16A attaches to soluble tumor-targeting mAbs and IgG fusion proteins. (A) Relative expression levels of CD16A and CD64/16A on NK92 cells were determined by cell staining with anti-CD16 and anti-CD64 mAbs (black bars), respectively, or an isotype-matched negative control antibody (gray bars). The bar graph shows mean fluorescence intensity (MFI) ± SD of three independent experiments. Representative flow cytometric data are shown in the histogram overlay. The dashed line histogram shows CD64 staining of NK92-CD64/16A cells, the orange-filled histogram shows CD16A staining of NK92-CD16A cells, and the green-filled histogram shows isotype control antibody staining of the NK92-CD16A cells. (B) NK92-CD16A and NK92-CD64/16A cells were incubated with or without trastuzumab (5 μg/ml) for 2 h at 37°C, washed, stained with a fluorophore-conjugated anti-human secondary antibody, and analyzed by flow cytometry. Data is representative of at least 3 independent experiments. (C) NK92-CD64/16A cells were incubated with cetuximab or rituximab (5 μg/ml for each), washed, and then stained with a fluorophore-conjugated anti-human secondary antibody. Control represents cells stained with the anti-human secondary antibody only. NK92-CD64/16A cells were also incubated with L-selectin/Fc (5 μg/ml), washed, and then stained with a fluorophore-conjugated anti-L-selectin mAb. NK92 cells lack expression of endogenous L-selectin (data not shown). All staining was analyzed by flow cytometry. Data shown are representative of 3 independent experiments. (D) NK92-CD16A and NK92-CD64/16A cells were incubated in the presence or absence of trastuzumab (5μg/ml), washed, and exposed to SKOV-3 cells (8 × 103) at the indicated E:T cell ratios for 2 h at 37°C. Data is shown as mean ± SD of 3 independent experiments. Statistical significance is indicated as **p < 0.01, ***p < 0.001. bd = below detection, (i.e., < spontaneous release by negative control cells). (E) NK92-CD16A and NK92-CD64/16A cells were incubated with SKOV-3 cells (E:T = 10:1; 8 × 104 cells to 8 × 103 cells) in the presence or absence of trastuzumab (5 μg/ml), as indicated, for 2 h at 37°C. Data is shown as mean ± SD of 3 independent experiments. Statistical significance is indicated as **p < 0.01.
Figure 4
Figure 4
Generation of iNK cells expressing CD64/CD16A. iPSCs were transduced to stably express CD64/16A, differentiated into NK cells, and then expanded using K562-mbIL21-41BBL feeder cells, as described in the Materials and Methods. iNK-CD64/16A cells and freshly isolated peripheral blood (PB) NK cells were stained for CD56, CD3 and various inhibitory and activating receptors, as indicated. CD64/16A expression was determined by staining the cells with an anti-CD64 mAb. For the iNK-CD64/16A cells, representative data from at least three independent experiments are shown. For the PB NK cells, three separate blood donors were examined and data from one donor is shown.
Figure 5
Figure 5
iNK-CD64/16A cells show enhanced ADCC compared to iNK-pKT2 control cells. (A) iNK cells derived from empty vector (iNK-pKT2) or CD64/16A (iNK-CD64/16A) transduced iPSCs were stained for CD56, CD64, and CD16A, as indicated. The percentage of CD56+ cells positively staining for CD16A or CD64 are indicated in the top right quadrant. (B) iNK-pKT2 or iNK-CD64/16A cells were incubated with SKOV-3 cells (E:T = 10:1; 8 × 104 cells to 8 × 103 cells) in the presence or absence of trastuzumab (5μg/ml), the function blocking anti-CD16 mAb 3G8 (5 μg/ml), and the function blocking anti-CD64 mAb 10.1 (5 μg/ml), as indicated, for 2 h at 37°C. Data is shown as mean ± SD of three independent experiments. Statistical significance is indicated as ***p < 0.001; ****p < 0.0001. (C) iNK-pKT2 and iNK-CD64/16A cells were incubated in the presence or absence of trastuzumab (5 μg/ml), washed, and exposed to SKOV-3 cells (E:T = 10:1; 8 × 104 cells to 8 × 103 cells) for 2 h at 37°C. Data is shown as mean ± SD of three independent experiments. Statistical significance is indicated as ***p < 0.001.
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