Targeting sinonasal undifferentiated carcinoma with a combinatory immunotherapy approach

Abstract

Purpose: Sinonasal undifferentiated carcinoma (SNUC) is a rare, aggressive malignancy of the sinonasal cavity with poor prognosis and limited treatment options. To investigate the potential for SNUC sensitivity to combinatory immunotherapy, we performed in vitro studies with SNUC cell lines and used multi-spectral immunofluorescence to characterize the in vivo patient SNUC tumor immune microenvironment (TIME).

Experimental design: Human-derived SNUC cell lines were used for in vitro studies of tumor cell susceptibility to natural killer (NK) cell-based immunotherapeutic strategies. Tumor samples from 14 treatment naïve SNUC patients were examined via multi-spectral immunofluorescence and clinical correlations assessed.

Results: Anti-PD-L1 blockade enhanced NK cell lysis of SNUC cell lines ∼5.4 fold (P ≤ 0.0001). This effect was blocked by a CD16 neutralizing antibody demonstrating activity through an antibody-dependent cellular cytotoxicity (ADCC) mediated pathway. ADCC-dependent lysis of SNUC cells was further enhanced by upregulation of PD-L1 on tumor cells by exogenous interferon-gamma (IFN-γ) administration or interleukin-15 (IL-15) stimulated IFN-γ release from NK cells. Combination treatment with anti-PD-L1 blockade and IL-15 superagonism enhanced NK-cell killing of SNUC cells 9.6-fold (P ≤ 0.0001). Untreated SNUC patient tumor samples were found to have an NK cell infiltrate and PD-L1⁺ tumor cells at a median of 5.4 cells per mm². A striking 55.7-fold increase in CK^low tumor cell/NK cell interactions was observed in patients without disease recurrence after treatment (P = 0.022). Patients with higher CD3⁺CD8⁺ in the stroma had a significantly improved 5-year overall survival (P = 0.0029) and a significant increase in CK^low tumor cell/CD8⁺ cytotoxic T cell interactions was noted in long-term survivors (P = 0.0225).

Conclusion: These data provide the pre-clinical rationale for ongoing investigation into combinatory immunotherapy approaches for SNUC.

Keywords: Antibody-dependent cellular cytotoxicity; IL-15; Immune microenvironment; Immunotherapy; Natural killer cells; Sinonasal undifferentiated carcinoma.

Fig. 1

SNUC cell lines are modestly susceptible to lysis by healthy donor natural killer (NK) cells at baseline, and cytotoxicity is enhanced by IFNγ-mediated PD-L1 upregulation and ligand targeting with N-601 through an ADCC-dependent mechanism. A. SNUC cell lines were used as targets for healthy donor NK cells versus non-treated (NT) controls. B. N-601 enhances NK cell killing of SNUC cells through antibody-dependent cellular cytotoxicity ADCC. N-601-mediated ADCC assays were performed by co-incubating SNUC cells with N-601 or isotype control antibody (Isotype Ab) prior to exposure to healthy donor NK cells (left). NK cells were treated with anti-CD-16 antibody for ADCC blocking experiments where indicated (right). C. IFNγ-treated SNUC cells show increased sensitivity to NK-cell lysis via N-601-mediated ADCC. SNUC cells were treated with 20 ng/mL of IFNγ or untreated as controls for 24 h prior to co-culture with N-601 or isotype control antibody (Isotype Ab). SNUC cells were used as targets for healthy donor NK cells in ¹¹¹In-release killing assays. NK cells were treated with anti-CD-16 antibody for ADCC blocking experiments where indicated. E:T ratios are 10:1. **P ≤ 0.01, ***P ≤ 0.001.

Fig. 2

N-803-activated NK cells show enhanced killing of SNUC cells, and combinatory treatment with N-803 and N-601 further enhances cytotoxicity. N-601-mediated ADCC assays were performed by co-incubating SNUC cells with N-601 or isotype control antibody. Combination treatment assays were performed by co-incubating SNUC cells with N-601 or isotype control antibody and treating NK cells with N-803. E:T ratios are 10:1. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 3

N-803 stimulates IFNy production from NK cells, which upregulates PD-L1 expression on SNUC cells and increases their sensitivity to N-601-mediated NK cell lysis. A. Healthy donor NK cells were cultured in 100 ng immobilized N-803 for 24 h or left untreated as controls. ELISA was used to quantify IFNy secretion within the supernatants of each NK cell culture. B. SNUC cells from the MDA8788–6 line were then cultured for 24 h in supernatant harvested from the N-803-treated NK cells, non-treated control supernatant, or in 20 ng/mL of IFNy as a positive control. SNUC cells were analyzed by flow cytometry for percent positive cells and MFI. C. SNUC cells from part B were used as targets for NK cell-mediated lysis via ¹¹¹In-release assay. SNUC cells were treated with N-601 or isotype control antibody, and combination treatment assays were performed by treating SNUC cells with N-601 or isotype control antibody and treating NK cells with N-803. E:T ratios are 10:1. **P ≤ 0.01, ****P ≤ 0.0001.

Fig. 4

PD-L1 t-haNKs induce lysis of SNUC cell lines in a PD-L1 dependent manner. SNUC cell lines were used as targets for PD-L1 t-haNK cells in ¹¹¹In-release killing assays. SNUC cells were co-cultured in 20 ng/mL of IFNγ or left untreated as controls. E:T ratios are 10:1. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001.

Fig. 5

The tumor immune microenvironment analysis in SNUC tumor samples. A. Quantification of NK cells per mm² in 14 SNUC tumor samples. B. Increased interactions between CK^low tumor cells and NK cells in the tumor parenchyma are associated with no disease recurrence C. Increased interactions between CK^low tumor cells and NK cells in stroma are associated with no disease recurrence. D. Quantification of CK⁺PD-L1⁺, CD3⁺CD8⁺ and CD68⁺ cells per mm² in 14 SNUC tumor samples. E. Patients with high CD3⁺CD8⁺ in stroma show significantly better 5-year overall survival rate (P = 0.0029). F. High CK^low cells in tumor and CD8⁺ in stroma interactions are associated with longer term survival. *P ≤ 0.05.