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Purpose: Antibodies against the lymphocyte PD-1 (aPD-1) receptor are cornerstone agents for advanced non–small cell lung cancer (NSCLC), based on their ability to restore the exhausted antitumor immune response. Our study reports a novel, lymphocyte-independent, therapeutic activity of aPD-1 against NSCLC, blocking the tumor-intrinsic PD-1 receptors on chemoresistant cells. Experimental Design: PD-1 in NSCLC cells was explored in vitro at baseline, including stem-like pneumospheres, and following treatment with cisplatin both at transcriptional and protein levels. PD-1 signaling and RNA sequencing were assessed. The lymphocyte-independent antitumor activity of aPD-1 was explored in vitro, by PD-1 blockade and stimulation with soluble ligand (PD-L1s), and in vivo within NSCLC xenograft models. Results: We showed the existence of PD-1+ NSCLC cell subsets in cell lines and large in silico datasets (Cancer Cell Line Encyclopedia and The Cancer Genome Atlas). Cisplatin significantly increased PD-1 expression on chemo-surviving NSCLC cells (2.5-fold P = 0.0014), while the sequential treatment with anti–PD-1 Ab impaired their recovery after chemotherapy. PD-1 was found to be associated with tumor stemness features. PD-1 expression was enhanced in NSCLC stem-like pneumospheres (P < 0.0001), significantly promoted by stimulation with soluble PD-L1 (+27% ± 4, P < 0.0001) and inhibited by PD-1 blockade (−30% ± 3, P < 0.0001). The intravenous monotherapy with anti–PD-1 significantly inhibited tumor growth of NSCLC xenografts in immunodeficient mice, without the contribution of the immune system, and delayed the occurrence of chemoresistance when combined with cisplatin. Conclusions: We report first evidence of a novel lymphocyte-independent activity of anti–PD-1 antibodies in NSCLC, capable of inhibiting chemo-surviving NSCLC cells and exploitable to contrast disease relapses following chemotherapy. See related commentary by Augustin et al., p. 505

PD-L1/2 and phospho-p38 MAPK expression in NSCLC cells. Tumor membrane expression of PD-L1 and PD-L2 in NSCLC cell lines was intensely enhanced by treatment with Interferon γ (n=9, PD-L1 p=0.02, PD-L2 p

Treatment with PD-L1s is associated with the up-regulation of expression of key protein coding genes involved in processes of tumorigenesis, EMT, tumor aggressiveness and chemoresistance. Heatmap showing z-scaled DESeq2-normalized expression values of the 14 differentially expressed genes between PD-L1s and untreated cells. Right annotation indicates the mean DESeq2-normalized gene expression values. Top annotations depict the pneumosphere of origin and the treatment. Hierarchical clustering of genes and samples is shown on the left and top, respectively (A). Volcano plot showing the log2 fold change (log2FC) and adjusted p-value values of all genes between PD-L1s and untreated samples. Differentially expressed genes are depicted in red (B).

Double positive OCT3/4-PD1 subsets in the bulk NSCLC cells after cisplatin treatment. OCT3/4 expression preferentially co-localized on PD-1+ NSCLC cells as compared with the PD-1- counterpart (14% vs 0.1%). Representative flow cytometry histograms of OCT3/4 expression are reported in figure.

Chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR Ts) produced impressive clinical results against selected hematological malignancies, but the extension of CAR T cell therapy to the challenging field of solid tumors has not, so far, replicated similar clinical outcomes. Many efforts are currently dedicated to improve the efficacy and safety of CAR-based adoptive immunotherapies, including application against solid tumors. A promising approach is CAR engineering of immune effectors different from αβT lymphocytes. Herein we reviewed biological features, therapeutic potential, and safety of alternative effectors to conventional CAR T cells: γδT, natural killer (NK), NKT, or cytokine-induced killer (CIK) cells. The intrinsic CAR-independent antitumor activities, safety profile, and ex vivo expansibility of these alternative immune effectors may favorably contribute to the clinical development of CAR strategies. The proper biological features of innate immune response effectors may represent an added value in tumor settings with heterogeneous CAR target expression, limiting the risk of tumor clonal escape. All these properties bring out CAR engineering of alternative immune effectors as a promising integrative option to be explored in future clinical studies.

Purpose: Aim of this study is to explore the intrinsic expression, functional role and therapeutic modulation of PD-1 receptor in Non Small Cell Lung Cancer (NSCLC) cells. We previously reported (Sanlorenzo M et al. Clinical Cancer Res 2018) that PD-1+ melanoma cells may sustain disease relapse following treatment with BRAF/MEK inhibitors. We hypothesized that PD-1+ tumor cells may characterize a “stem-like” compartment also in NSCLC, sustaining chemo-resistance and disease relapse with potential therapeutic implications. Experimental procedures: The expression of PD-1 by NSCLC cells was explored by flow cytometry, western blot (WB) and RT-PCR. Its presence and role in lung cancer stem cells (CSC) was explored by sphere formation essays. CSC were further visualized by tumor-engineering with a lentiviral CSC-detector vector (LV-CSC) encoding eGFP under control of the OCT4 stem gene promoter. Selective PD-1 blockade and PD-1 stimulation with soluble ligand (PD-L1) were used to study the functional role and therapeutic modulation of intrinsic PD-1 in NSCLC. Results: PD-1 is intrinsically expressed by a small subset of NSCLC cells with stemness features. We found PD-1 consistently expressed on the membrane of a small tumor cell fraction (2% ± 0.3) within 6 NSCLC cell lines (H1975, EBC-1, H23, H820, HCC827), including a primary patient-derived NSCLC culture (SL1). Data were confirmed by RT-PCR and WB. Viable PD-1+ tumor cells were significantly enriched in NSCLC spheres generated in stem conditioned cultures, compared with the monolayer controls (10% [4-36] vs 2% [1-5] P Conclusions: PD-1 is intrinsically expressed by NSCLC cells with stemness features and mediates pro-tumorigenic activity. PD-1+ NSCLC cells are enriched after chemotherapy and may be inhibited by selective PD-1 blockade. We report a new, intrinsic, expression pattern of PD-1 in NSCLC, providing rationale to explore a lymphocyte-independent activity of anti-PD1 antibodies. Citation Format: Ramona Rotolo, Valeria Leuci, Chiara Donini, Martina Sanlorenzo, Igor Vujic, Giovanni Medico, Francesca Vita, Loretta Gammaitoni, Luisella Righi, Chiara Riganti, Elisa Vigna, Lorenzo D'Ambrosio, Giovanni Grignani, Giorgio V. Scagliotti, Silvia Novello, Massimo Aglietta, Dario Sangiolo. PD-1 is intrinsically expressed by lung cancer cells with stemness features inhibited by PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3234.