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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.

Peritoneal carcinomatosis (PC) from ovarian cancer (OC) has poor prognosis and is in need for innovative therapies. Such clinical condition displays a composite structure with floating cell aggregates within ascites and solid-like masses invading the peritoneum. A deep understanding of the dynamics is thus crucial to propose more effective treatments. Chimeric antigen receptor (CAR)-based immunotherapies are emerging as promising approaches, and ex vivo expanded cytokine-induced killer lymphocytes (CIK), intrinsically endowed with HLA-independent antitumor activity, are a valid platform to be exploited against solid tumors. Appropriate preclinical models, recapitulating the clinical complexity, are needed in the perspective of clinical translation. Here we explored the activity of mesothelin (MSLN) CAR-redirected CIK (CAR.CIK) against advanced OC, by developing 3D models resembling PC structural complexity. CAR.CIK were generated from patients’ PBMC (n=11), engineered with 2nd generation MSLN-CAR with 4-1BB costimulation. As tumor targets we employed and characterized a panel of ascites-derived OC cell lines (aOC). Anti-aOC CAR.CIK killing ability was evaluated mostly by flow cytometry. CAR.CIK were successfully generated with a mean CAR expression of 42%. MSLN was highly expressed (>75%) in 5/6 aOC. CAR.CIK effectively killed tumor targets in standard 2D assays, even at very low effector/target ratios (E/T), with significant improvement compared to unmodified CIK (NTD.CIK): 78% vs 36%, 46% vs 15% respectively at 5:1 and 1:2 E/T (p In order to recapitulate the clinical complexity we generated 3D aOC spheroids, either floating in liquid medium or embedded in a 3D hydrogel and co-cultured with CAR.CIK. To measure CAR.CIK activity we employed live-imaging and image processing. CAR.CIK were found to localize faster than NTD.CIK in both liquid and solid settings. Recruitment kinetics of CAR.CIK on aOC aggregates in liquid was significantly faster in shaking cultures, reminiscent of spontaneous patient movement, than in absence of flow. Even in the 3D solid setting, CAR.CIK were functionally more efficient and localized faster on aOC spheroids than NTD.CIK. Our data indicate that MSLN-CAR.CIK are effective against aOC in both 2D and 3D settings. Kinetics measurements in 3D show that recruitment in liquid requires shorter times when coupled to fluid flow. The solid setting is instead characterized by slower kinetics but CAR.CIK anti-tumor activity remains comparable to the liquid condition. The kinetics of recruitment should therefore be considered when assessing CAR.CIK killing. Our findings provide reliable translational bases for clinical studies to explore intraperitoneal cellular immunotherapy with CAR.CIK against OC PC. Citation Format: Federica Galvagno, Valeria Leuci, Chiara Donini, Annamaria Massa, Ramona Rotolo, Sonia Capellero, Alessia Proment, Letizia Vitali, Valentina Tuninetti, Martina Olivero, Elisa Vigna, Giorgio Valabrega, Luca Primo, Alberto Puliafito, Dario Sangiolo. Anti-mesothelin CAR.CIK lymphocytes are effective against ovarian cancer in 3D models of peritoneal carcinomatosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3194.