Immunotherapy utilizing the combination of natural killer- and antibody dependent cellular cytotoxicity (ADCC)-mediating agents with poly (ADP-ribose) polymerase (PARP) inhibition

Abstract

Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) prevent single-stranded DNA repair. Olaparib is a PARPi approved for the treatment of BRCA mutant ovarian and breast carcinoma. Emerging clinical data suggest a benefit of combining olaparib with immunotherapy in prostate cancer patients both with and without somatic BRCA mutations.

Methods: We examined if olaparib, when combined with IgG₁ antibody-dependent cellular cytotoxicity (ADCC)-mediating monoclonal antibodies (mAbs) cetuximab (anti-EGFR), or avelumab (anti-PD-L1), would increase tumor cell sensitivity to killing by natural killer (NK) cells independently of BRCA status or mAb target upregulation. BRCA mutant and BRCA wildtype (WT) prostate carcinoma cell lines were pretreated with olaparib and then exposed to NK cells in the presence or absence of cetuximab or avelumab.

Results: NK-mediated killing was significantly increased in both cell lines and was further increased using the ADCC-mediating mAbs. Pre-exposure of NK cells to recombinant IL-15/IL-15Rα further increased the lysis of olaparib treated tumor cells. In addition, olaparib treated tumor cells were killed to a significantly greater degree by engineered high-affinity NK cells (haNK). We show here for the first time that (a) olaparib significantly increased tumor cell sensitivity to NK killing and ADCC in both BRCA WT and BRCA mutant prostate carcinoma cells, independent of PD-L1 or EGFR modulation; (b) mechanistically, treatment with olaparib upregulated death receptor TRAIL-R2; and (c) olaparib significantly enhanced NK killing of additional tumor types, including breast, non-small cell lung carcinoma, and chordoma.

Conclusions: These studies support the combined use of NK- and ADCC-mediating agents with correctly timed PARP inhibition.

Keywords: ADCC; BRCA; PARP inhibitors; Prostate carcinoma; TRAIL.

Fig. 1

NK-mediated lysis of prostate carcinoma cells is increased following exposure to olaparib. a Real-time impedance-based cell analysis of BRCA mutant prostate carcinoma (22RV1) cells in the presence or absence of olaparib (ola) and NK. b Lysis of BRCA mutant cells treated with or without olaparib and NK compared to control at 12 h and 36 h. c Real-time impedance-based cell analysis of BRCA WT prostate carcinoma (DU145) cells in the presence or absence of olaparib and NK. d Lysis of BRCA WT cells treated with olaparib and NK compared to control at 12 h and 36 h. These experiments were performed with four different human NK donors with similar results. p < 0.01**, p < 0.001***, p < 0.0001****

Fig. 2

NK-mediated lysis of BRCA mutant prostate carcinoma cells treated with olaparib is further increased by the addition of an ADCC-mediating antibody. a Real-time impedance-based cell analysis of BRCA mutant (mut) prostate carcinoma (22RV1) cells in the presence or absence of olaparib (ola) and human NK cells, treated with either cetuximab (cet) or isotype control. b 22RV1 cells lysed with or without olaparib pretreatment and NK cells, treated with either cetuximab or isotype control at 12 h and 36 h. This experiment was performed with four different human NK donors with similar results. p < 0.001***, p < 0.0001****

Fig. 3

NK-mediated lysis of BRCA wildtype prostate carcinoma cells treated with olaparib is further increased by the addition of ADCC-mediating antibodies. a Real-time impedance-based assay of BRCA WT prostate carcinoma (DU145) cells in the presence or absence of olaparib (ola), human NK cells, and mAbs. b Lysis of BRCA WT cells with or without olaparib pretreatment and NK cells, and with cetuximab (cet) or isotype control at 12 h and 36 h. c Lysis of DU145 cells with or without 24 h pretreatment with olaparib and NK cells, with either avelumab (ave) or isotype control at 12 h and 36 h. These experiments were performed with four different human NK donors with similar results. p < 0.01**, p < 0.0001****

Fig. 4

Olaparib treatment enhances ADCC using both cetuximab and avelumab without modulation of mAb targets EGFR and PD-L1. a Treatment with cetuximab (cet) significantly increased NK-induced lysis of olaparib (ola)-treated BRCA mutant prostate carcinoma (22RV1) cells at 12 h. The addition of anti-CD16 antibody neutralized this increase, confirming that the increased lysis is attributable to ADCC. b STING is not expressed in 22RV1 either before or after olaparib treatment. c Olaparib treatment did not result in significant modulation of EGFR expression on 22RV1 cells as measured by flow cytometry. d Treatment with cetuximab increased NK-induced lysis of olaparib-treated BRCA WT prostate carcinoma cells (DU145) cells. Role of anti-CD16 antibody on increased lysis attributable to ADCC. e The PD-L1+ cell line DU145 also underwent NK-induced ADCC in the presence of the anti-PD-L1 antibody avelumab (ave). Lysis of DU145 cells after 12 h in the presence or absence of olaparib and NK, treated with either avelumab or isotype control is shown. f STING was upregulated in DU145 following exposure to olaparib. g Olaparib treatment did not result in significant modulation of EGFR expression in DU145 cells as measured by flow cytometry. h Olaparib treatment did not result in significant modulation of PD-L1 expression in DU145 cells as measured by flow cytometry. These experiments were performed twice with similar results. p < 0.05*, p < 0.0001****

Fig. 5

Olaparib treatment induces increased expression of gene TNFRSF10B (TRAIL-R2) in both BRCA mutant and wildtype prostate carcinoma. a Heatmap demonstrating genes modulated by at least 3-fold after olaparib treatment as identified by the NanoString nCounter® PanCancer Pathways Panel. TNFRSF10B (TRAIL-R2) was upregulated in both cell lines (by 2-fold in 22RV1 and by 20% in DU145). b Summary of modulated genes identified in NanoString analysis. c Summary of modulated genes identified in Apoptosis RT² Profiler PCR Array with fold-change denoted in parentheses. d STRING network of protein-protein interactions of a curated set of modulated genes identified by both NanoString and apoptotic array. TNFRSF10B (TRAIL-R2; red box) identified as the nexus of both sets

Fig. 6

TRAIL-R2 is functionally upregulated upon exposure to olaparib and leads to increased caspase cascade activation. a Representative image demonstrated NK cell lysing olaparib-treated BRCA WT target cell by activating caspase cascade and a representative graph demonstrating increased caspase activation as identified by a FLICA reagent following administration of olaparib in both BRCA WT and BRCA mutant cells. b Expression of TRAIL-R2 on 22RV1 was upregulated from 36% (MFI 104) to 65% (MFI 151) following a 48 h treatment with olaparib. c BRCA mutant prostate carcinoma (22RV1) cell lysis with or without olaparib (ola) and KillerTRAIL antibodies after 36 h. d Expression of TRAIL-R2 on DU145 was upregulated from 3% (MFI 28) to 96% (MFI 103). e BRCA WT prostate carcinoma (DU145) cell lysis with or without olaparib and KillerTRAIL antibodies after 36 h. f Expression of TRAIL-R2 on Wild-Type and TRAIL-R2 CRISPR knockout DU145 cells. g Functional confirmation of the involvement of TRAIL-R2. DU145 and TRAIL-R2 CRISPR knockout DU145 cell lysis with or without olaparib and NK cells after 36 h. Depicted is fold-change in percent lysis. These experiments were performed twice with similar results. p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001***

Fig. 7

Lysis of target cells by natural killer (NK) cells treated with N-803 or high-affinity NK cells (haNK) can be further increased with the addition of olaparib. a Lysis of BRCA wildtype prostate carcinoma (DU145) cells at 12 h and 36 h with or without pretreatment with olaparib (ola) and NK cells incubated in the presence or absence of an IL-15/IL-15Rα superagonist (N-803). b Lysis of DU145 at 36 h by haNK cells in the presence or absence of olaparib and avelumab (ave). p < 0.05*, p < 0.01**, p < 0.0001****

Fig. 8

Olaparib increases NK-mediated target cell lysis and ADCC in a diverse set of tumors. A variety of tumor types demonstrated increased NK cell-mediated lysis as measured by real-time impedance. a Lysis of estrogen receptor positive (ER+) breast carcinoma (MCF7) in the presence or absence of olaparib and NK, treated with cetuximab or isotype control. b Lysis of non-small cell lung carcinoma (NSCLC) (H460) in the presence or absence of olaparib and NK, treated with cetuximab, avelumab (ave) or isotype control. c Lysis of triple negative breast carcinoma (SUM149) in the presence or absence of olaparib and NK, treated with cetuximab (cet) or isotype control. d Lysis of chordoma (Ch22) in the presence or absence of olaparib and NK, treated with cetuximab, avelumab or isotype control. p < 0.01**, p < 0.001***, p < 0.0001****