MICA/B-targeted antibody promotes NK cell-driven tumor immunity in patients with intrahepatic cholangiocarcinoma

Abstract

The major histocompatibility complex-class I chain related proteins A and B (MICA/B) is upregulated because of cellular stress and MICA/B shedding by cancer cells causes escape from NKG2D recognition favoring the emergence of cancers. Cholangiocarcinoma (CCA) is a relatively rare, though increasingly prevalent, primary liver cancer characterized by a late clinical presentation and a dismal prognosis. We explored the NKG2D-MICA/B axis in NK cells from 41 patients with intrahepatic cholangiocarcinoma (iCCA). The MICA/B-specific 7C6 mAb was used for ex vivo antibody-dependent cytotoxicity (ADCC) experiments using circulating, non tumor liver- and tumor-infiltrating NK cells against the HuCCT-1 cell line and patient-derived primary iCCA cells as targets. MICA/B were more expressed in iCCA than in non-tumoral tissue, MICA transcription being higher in moderately-differentiated compared with poorly-differentiated cancer. Serum MICA was elevated in iCCA patients in line with higher expression of ADAM10 and ADAM17 that are responsible for proteolytic release of MICA/B from tumor. Addition of 7C6 significantly boosted peripheral, liver- and tumor-infiltrating-NK cell degranulation and IFNγ production toward MICA/B-expressing established cell lines and autologous iCCA patient target cells. Our data show that anti-MICA/B drives NK cell anti-tumor activity, and provide preclinical evidence in support of 7C6 as a potential immunotherapeutic tool for iCCA.

Keywords: ADCC; Natural killer cells; immunotherapy; innate immunity; liver cancer.

Figure 1.

MICA/B proteins are expressed in iCCA tissues. A): mRNA MICA expression on iCCA tissues along with matched NT specimens (n = 13). Lower panel showed mRNA MICA expression in tissues stratified according to tumor grade. G2, moderately-differentiated tumor; G3, poorly-differentiated tumor. Paired and unpaired t tests were used to compare data. A representative immunohistochemical staining of MICA/B protein expression on iCCA tissues (b-d), NT (e) and HL specimens (f). In iCCA specimens MICA/B shows as brown cytoplasmic granules while it shows as a diffuse faint staining in NT tissues and in HL.

Figure 2.

Serum MICA levels and tissue ADAM10/ADAM17 proteases are elevated in iCCA patients A): Serum MICA concentrations in iCCA patients (n = 35) and HC (n = 46). The Mann-Whitney U test was used to compare data. Middle bars represent the median values; box plots are 25% and 75% percentiles; and whiskers are minimum and maximum values. B): mRNA/ADAM10 (left panel) and mRNA/ADAM17 (right panel) ratios in iCCA tissues and in matched NT specimens (n = 13). C): Analysis of transcription in the same tissues of panel B stratified according to tumor grade. G2, moderately-differentiated tumor (n = 4); G3, poorly-differentiated tumor (n = 9). The non-parametric paired Wilcoxon test and unpaired Mann-Whitney U test were used to compare data. D, E): Representative immunohistochemical staining of ADAM10 (d) and ADAM17 (e) protein expression on iCCA tissues, NT and HL specimens.

Figure 3.

Circulating NK cells from iCCA patients displayed an altered frequency and phenotype. A): frequencies of circulating CD3-CD56+ NK cells, CD56^dim and CD56^bright subsets in iCCA patients (n = 23) and HC (n = 16). C, E): frequencies and expression (MFI) of NKG2D receptor (iCCA, n = 23; HC, n = 16) and cytotoxic GrB granules (iCCA, n = 23; HC, n = 16) on NK cells of iCCA patients and HC. The non-parametric unpaired t test was used to compare data. B, D, F): Representative dot plots and histograms of a HC and an iCCA to show NK, NKG2D and GrB distribution and expression. Middle bars represent median values; box plots are 25% and 75% percentiles; and whiskers are minimum and maximum values.

Figure 4.

Reduced expression of NKG2D, DNAM-1 and CD69 on tumor-infiltrating NK cells from iCCA patients. A): Frequency of total CD3-CD56+ NK cells and of the CD16+ and CD16- subsets in LIL and TIL of iCCA patients (n = 10). Frequency and expression (MFI) of NKG2D (c), DNAM-1 (d) and CD69 (e) on LIL-and TIL-NK cell of iCCA patients. The Wilcoxon matched-pairs signed rank test was used to compare paired data. B, F, G and H): Representative dot plots of the frequency of total NKG2D+, DNAM-1+ and CD69+ NK cell distribution and expression (MFI) on LIL- and TIL-NK of an iCCA patient. Middle bars represent the median values; box plots are 25% and 75% percentiles; and whiskers are minimum and maximum values.

Figure 5.

Reduced expression of activating receptors on TIL-NK compared to LIL-NK after IL15 stimulation. A) Frequency of total CD3-CD56+ NK cells and of the CD16+ and CD16- subsets on LIL and TIL from iCCA patients (n = 10). Frequency and expression (MFI) of NKG2D (b), DNAM-1 (c), CD69 (d), TRAIL (e), NKp30 (f) and NKp46 (g) on LIL-and TIL-NK cell of iCCA patients. The Wilcoxon matched-pairs signed rank test was used to compare paired data. Middle bars represent the median values; box plots are 25% and 75% percentiles; and whiskers are minimum and maximum values.

Figure 6.

Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against the HuCCT-1 cell line. A): peripheral NK cell degranulation, evaluated as frequency of CD107a+ NK cells, in iCCA patients (n = 13) and HC (n = 16) in the presence of 7C6 mAb or IgG1-Fc. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56+ CD107a+ NK cells in a HC and a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): the proportion of circulating IFNγ+ NK cells in patients (n = 10) and HC (n = 11) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, the parametric t test and the non-parametric Wilcoxon t test were used. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Figure 7.

Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against patient-derived iCCA cell lines. A): peripheral NK cell degranulation, evaluated as CD107a+NK frequency, in iCCA patients (n = 12) and HC (n = 8) in the presence of 7C6 mAb or IgG1-Fc using patient-derived primary tumor cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56+ CD107a+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): proportion of circulating IFNγ+NK cells in patients (n = 10) and in HC (n = 8) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, we used the parametric t test and the non-parametric Wilcoxon t test. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ+NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Figure 8.

7C6 mAb enhances the anti-tumor effect of liver- and tumor-infiltrating NK cells in iCCA patients. A): Frequency of degranulating CD107a+NK cells in LIL (n = 13) and TIL (n = 10) of iCCA patients in the presence of anti-MICA/B 7C6 mAb or IgG1-Fc using autologous tumor-derived cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): representative dot plots showing the frequency of CD3-CD56+ CD107a+ LIL- and TIL-NK cells in the presence of 7C6 mAb or IgG1-Fc. C): proportion of IFNγ+ NK cells in LIL (n = 10) and TIL (n = 8) of iCCA patients in the presence of 7C6 mAb compared with IgG1-Fc using autologous tumor-derived cell lines as targets. The parametric t test and non-parametric Wilcoxon t test were used to compare paired data. The parametric t test was used to compare unpaired data.

Figure 9.

Cytotoxicity assay of HC PBMC, patient PBMC, LIL and TIL cells. A, B): Frequency of CFSE+LIVE/DEAD (LD)+ HuCCT-1 cell line targets when HC PBMC (n = 5), patient PBMC (n = 10), LIL (n = 8) and TIL (n = 5) were used as effector cells in the presence of 7C6 mAb and isotype control (IgG1). The parametric paired t tests were used to compare data. C, D): Frequency of CFSE+LD+ patient-derived cell line targets when HC PBMC (n = 5), patient PBMC (n = 8), LIL (n = 8) and TIL (n = 4) were used as effectors in the presence of 7C6 and isotype control. The parametric paired t test was used to compare data in panel C. The non-parametric Wilcoxon t test was used to compare paired data in panel D. Target cell death was determined as frequency of CFSE+LD+ cells.

Figure 10.

Increased MICA/B expression on tumor cells after 7C6 mAb pretreatment. A): MICA/B expression on HuCCT-1 cells after treatment with 7C6 mAb and isotype control. B): frequency of CD107a+ NK cells from HC in the presence of 7C6 mAb using 7C6- and isotype-pretreated HuCCT-1 cells as target. The parametric paired t tests were used to compare data. C): representative dot plots showing the frequency of CD3-CD56+ CD107a+ NK cells shown in panel b. D): MICA/B expression (MFI) on patient-derived tumor cell lines (n = 10) after treatment with 7C6 mAb and isotype control. The non-parametric Wilcoxon t test was used to compare data. E): representative histograms showing MICA/B expression on patient-derived tumor cell lines illustrated in panel D.