Avelumab, an IgG1 anti-PD-L1 Immune Checkpoint Inhibitor, Triggers NK Cell-Mediated Cytotoxicity and Cytokine Production Against Triple Negative Breast Cancer Cells

Abstract

The standard treatment for Triple Negative Breast Cancer (TNBC) patients is cytotoxic chemotherapy, but it is restricted since the duration of response is usually short. Blocking the PD-1/PD-L1 pathway through monoclonal antibodies (mAbs) appears to be a promising therapeutic strategy for TNBC patients. Avelumab is a human IgG1 anti-PD-L1 mAb being tested in clinical trials that may also trigger antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells as an additional antitumor activity. In the present work, we studied in vitro Avelumab-mediated ADCC against a panel of TNBC cells with different PD-L1 expression using peripheral blood mononuclear cells (PBMC) or purified NK cells from healthy donors. We determined that Avelumab significantly enhanced NK-cell mediated cytotoxicity against TNBC cells and that tumor cells expressing higher levels of PD-L1 were more sensitive to Avelumab-mediated ADCC. IFN-γ treatment upregulated PD-L1 expression in tumor cells but had a variable impact on Avelumab-mediated ADCC, which could be related to the simultaneous effect of IFN-γ on the expression of NK cell ligands. Moreover, IL-2 and IL-15 stimulation of NK cells enhanced Avelumab-triggered cytokine production and degranulation along with increased lytic activity against tumor cells. Improving the treatment of TNBC remains still a considerable challenge. This in vitro study suggests that Avelumab-mediated ADCC, independently of the blockade of the PD-1/PD-L1 pathway, could be a valuable mechanism for tumor cell elimination in TNBC. Avelumab combination with immunomodulators such as IL-15 or IL-2 could be taken into consideration to increase the therapeutic efficacy of Avelumab in TNBC.

Keywords: ADCC; Avelumab; IFN-γ; IL-15; IL-2; PD-L1; triple negative breast cancer (TNBC).

Figure 1

PD-L1 expression and Avelumab mediated-ADCC of TNBC cell lines (A) PD-L1 expression in five TNBC cell lines represented as normalized MFI (MFI of cells stained with specific mAb/ MFI of cells stained with isotype control) in the left, and percentage of positive cells with representative histograms in the right. Cells were analyzed by FACS after staining with APC anti-PDL1 (clone 29E.2A3). In all of the figures, cells with higher expression of PD-L1 are shown in orange and the ones with lower expression, in gray. (B) Percentage of lysis against the five TNBC cell lines coated with 1 μg/ml of isotype control (IgG1) or Avelumab, at 5:1 NK:BC ratio. (C) Percentage of lysis against BT-549 and MDA-MB-468 cells (low PD-L1 expression) coated with IgG1 or Avelumab, at 10:1 NK:BC ratio. In (B,C) PBMC from the same six donors were used as effector cells for all the cell lines; ^**p < 0.01 (paired t-test). (D) Percentage of degranulating NK cells (CD3⁻CD56⁺CD107a⁺) against MDA-MB-231 (high PD-L1 expression) and BT-549 (low PD-L1 expression) coated with IgG1 or Avelumab using PBMC from the same six donors (each symbol represents an individual donor). Basal degranulation without target cells is shown in light-blue symbols. Bars with different letters are statistically different,p < 0.05 (ANOVA). (E) Percentage of lysis against MDA-MB-231 cells coated with IgG1 or Avelumab using PBMC or purified NK cells as effector cells (n = 4); ^**p < 0.01 (paired t-test). (F) Percentage of lysis against MDA-MB-231 cells coated with IgG1, a mutant IgG1 anti-PD-L1 mAb that does not bind to human Fcγ receptors (Atezolizumab), or Avelumab using PBMC as effectors at 5:1 NK:BC ratio (n = 3). Bars with different letters are statistically different, p < 0.05 (ANOVA).

Figure 2

IFN-γ effect on PD-L1 and NK cell ligand expression and susceptibility to Avelumab-mediated ADCC of TNBC cell lines. TNBC cells lines were treated with 10 U/ml IFN-γ for 24 h or left untreated before being used in the assays. In all of the figures, cells with higher basal expression of PD-L1 are shown in orange and the ones with lower basal expression, in gray. Lysis of IFN-γ treated and untreated cancer cells coated with IgG1 or Avelumab was evaluated using PBMC as effector cells at 5:1 or 10:1 NK:BC ratios (n = 6–7): (A) Changes in lysis (Δ % Lysis) of treated compared to untreated cancer cells were quantified for each donor and results are shown as box plots; (B) Percentage of lysis against MDA-MB-231 cells at 5:1 NK:BC ratio; (C) Percentage of lysis against BT-549 cells at 5:1 NK:BC ratio; (D) Percentage of lysis against MDA-MB-468 cells at 10:1 NK:BC ratio. Bars with different letter are statistically different, p < 0.05 (ANOVA). (E) PD-L1 expression in IFN-γ treated and untreated TNBC cell lines represented as normalized MFI (MFI of cells stained with specific mAb/MFI of cells stained with isotype control). ^*p < 0.05; ^**p < 0.01 (paired t-test). (F) Heat-map representing basal expression of NK cell ligands in TNBC cell lines, showed as log₂ normalized MFI (left) and heat-map representing fold change in NK cell ligand expression after IFN-γ treatment of TNBC cell lines, showed as log₂ fold change in normalized MFI (right).

Figure 3

IL-2 and IL-15 effect on NK cell degranulation and cytokine production triggered by Avelumab coated TNBC cells. PBMC were stimulated ON with 1,000 U/ml IL-2 or 10 ng/ml IL-15, or left untreated, and then cultured without target cells or with MDA-MB-231 coated with IgG1 or Avelumab to study degranulation and cytokine production as described in Materials and Methods: (A) Percentage of NK cells that produced IFN-γ (CD3⁻CD56⁺IFN-γ⁺); (B) Percentage of NK cells that produced TNF-α (CD3⁻CD56⁺TNF-α⁺); (C) Percentage of NK cells that degranulated (CD3⁻CD56⁺CD107a⁺). Bars with different letter are statistically different, p < 0.05 (n = 6, ANOVA). (D) Area-Proportional Venn diagrams were used to represent the percentage of NK cells expressing IFN-γ, TNF-α, CD107a or co-expressing these markers against MDA-MB-231 cells coated with Avelumab. Effector cells were rested (left) or stimulated ON with IL-2 (middle) or IL-15 (left) before the assay.

Figure 4

IL-2 and IL-15 effect on Avelumab-mediated ADCC of TNBC cell lines. Cytotoxic assays were performed at 5:1 NK:BC ratio with PBMC un-stimulated or pre-stimulated ON with 1,000 U/ml IL-2 or 10 ng/ml IL-15: (A) Percentage of lysis against MDA-MB-231 cells (high basal PD-L1 expression) coated with IgG1 or Avelumab; (B) Percentage of lysis against MDA-MB-468 cells (low basal PD-L1 expression) coated with IgG1 or Avelumab; (C) Percentage of lysis against BT-549 cells (low basal PD-L1 expression) coated with IgG1 or Avelumab, un-treated (left) or pre-treated with IFN-γ (right). Bars with different letters are statistically different, p < 0.05 (n = 6, ANOVA).