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Background: Malignant pleural/peritoneal mesothelioma (MPM) is a rare, aggressive cancer with poor prognosis and high mortality of 65%-70% for pleural and 30% for peritoneal MPM. Patients who fail the standard therapy often survive less than 1 year, so it is urgent to develop new effective therapies for MPM patients. Chimeric antigen receptor (CAR)-T cells have been applied in MPM, but the efficacy was still limited due to immunosuppressive tumor microenvironment (TME). To overcome these obstacles, we developed armored CAR-T cells with nanobody targeting mesothelin (MSLN) and IFN-γ-activated secretion of PD-1 nanobody in a non-viral transposon system, named as BZDS1901. Preclinical studies have demonstrated cytotoxicity of the BZDS1901 in NCI-H226 lung/mesothelioma xenograft mouse model. To verify the safety and efficacy of BZDS1901, we conducted a single-arm, open label, dose-escalating clinical trials (NCT04503980, 05089266, 03615313) in solid tumors. Methods: Eligible patients were those who failed prior standard therapies with MSLN expression (≥50%) and PD-L1 positive in tumor specimen and voluntarily signed the informed consent. After apheresis and lymphodepletion with cyclophosphamide and fludarabine. BZDS1901 was administered intravenously in dose cohorts (1 × 106-2 × 107/kg) and the second infusion was given if no disease progression. After infusion, safety was evaluated during 28 days by the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0, efficacy was assessed by RECIST 1.1 or mRECIST with CT scan. Blood CAR copies were measured by qPCR, PD-1 nanobodies and cytokines by Meso Scale Discovery method, and T cell subtypes by flow cytometry. Patients’ progression-free survival (PFS) and overall survival (OS) were measured from the day of infusion to progression or death. Results: From July 20, 2020 to December 31, 2022, 11 MPM patients were enrolled and completed the assessment, while most patients received two infusions. BZDS1901 was safe, demonstrated by 54% grade 3 and 15% grade adverse events (AEs) that were hematological side effects due to lymphodepletion and reversible with supportive care. No on-target, off-tumor toxicity and dose-limiting toxicity were observed. All patients showed expansion of CAR-T cells and increased PD-1 nanobodies in circulation. CAR-T Cmax (cp/μg) copies number was 20062, and continually detectable in blood over 4 months. PD-1 Cmax (pg/ml) was 82841, and continually detectable in blood for up to 9 months. IFN- γ and IL-6 also increased at day 4 or Day 7. All patients obtained objective tumor response, one with complete response, six with partial response, and four with stable disease. The total objective response rate was 63.64%. All enrolled patients are still alive, and mPFS and mOS are not reached. The longest PFS was up to 26 months. Median follow-up was four months. Conclusions: PD-1 nanobody secreted and MSLN targeting CAR-T cells have demonstrated promising efficacy on MPM patients. Besides CAR-T direct tumor killing activity, secreted PD-1 nanobodies may provide additional clinical benefit by invigorating CAR-T from PD-L1 inhibition, activating TILs and relieving local immunosuppression. Citation Format: Zhuqing Liu, Yong Xia, Linlin Li, Yan Sun, Zhicai Lin, Lijie Rong, Zhongzheng Zhu, Zongchang Song, Hui Xue, Jianchun Duan, Shujing Shen, Jing Wang, Linjie Lv, Yaping Yang, Xue Tan, Liping Han, Wei Zhao, Jie Wang, Wenfeng Xu, Weimin Zhu, Zhong Li, Xingya Li, Jinxing Lou, Qing Xu, Qijun Qian. Non-viral mesothelin-targeted CAR-T cells armored with IFNg-induced secretion of PD-1 nanobody in treatment of malignant mesothelioma in phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT134.

e17535 Background: Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of cancer-related death among women. Most of patients often have refractory or relapse. We have developed a CAR T cell secreting PD-1 nanoantibodies and targeting mesothelin (αPD-1-mesoCAR-T) and conducted a phase I clinical trial for evaluation of safety and efficacy of the αPD-1-mesoCAR-T cells in gynecologic tumors. Methods: This study is a single-arm, open, dose-increasing clinical study and two infusions (interval 14-35 days). Patients with advanced gynecological cancer were enrolled when immunohistochemical analysis for positive mesothelin over 70% and failed second-line treatment in Mengchao Cancer Hospital affiliated to Shanghai University and Shanghai Tenth People's Hospital affiliated to Tongji University. All patients underwent lymphodepletion before infusion at a dose of 1 to 15x106/kg. CAR copies, nanobody levels, cytokines, T lymphocyte subtypes were determined before and after infusion. Results: a total of 7 patients were enrolled so far and completed the first evaluation. After treatment, most symptoms of the patients were alleviated, two patients (#2, #3) had their lesions decreased. By RECIST 1.1, all 7 patients’ diseases became stable, and the disease control rate was 100%. The PFS of #3 patient with stable disease (SD) was 5.3 months, the patient #2 was 4.1 months. The frequent adverse events were fever, fatigue, nausea and pruritus. No sever adverse events above grade 3 occurred. The number of CAR-T cells in the peripheral blood increased significantly during day 4-14 after the infusion, and the PD-1 nanoantibody levels and the contents of INF-γ、IL-6、IL-2 and MCP-1 also increased at day 1-4. Conclusions: The preliminary clinical study showed that the aPD-1-mesoCAR-T cells are safe for the treatment of gynecologic cancer and stabilized the diseases. More data of the treatment is warrantied to continually follow up these patients.

8559 Background: Malignant pleural/peritoneal mesothelioma (MPM) is an aggressive cancer characterized by poor therapeutic response and poor survival. Programmed cell death protein-1 (PD-1)-mediated immunosuppression is believed to be associated with T cell exhaustion and dysfunction in solid tumors. We generated chimeric antigen receptor (CAR-T) cells that can secret PD-1 nanobodies and target mesothelin (PD-1-mesoCAR-T). After identifying the anti-tumor activity and cytotoxicity of PD-1-mesoCAR-T cells by pharmacokinetics and pharmacodynamics, we conducted a proof-of-concept clinical trial in the Shanghai Tenth People's Hospital affiliated to Tongji University and the Mengchao Tumor Hospital affiliated to Shanghai University to evaluate the safety and efficacy of PD-1-mesoCAR-T cells in a dose-escalation study for malignant pleural/peritoneal mesothelioma. 6 patients received one or more infusions of the CAR-T cells with prior lymphodepletion and the longest survival is up to 18 months. Methods: This open-label, single-arm, multicenter, phase 1 study was enrolled patients with pathological diagnosis with malignant mesothelioma whose mesothelin expression in immunohistochemical samples was equal or greater than 50%. Immunohistochemical samples derived from surgical tissue, puncture tissue or pleural effusion. PD-1-mesoCAR-T cells were administered intravenously ranging 1×106−1.5×107/kg. Safety and efficacy were assessed in response-evaluable patients (ie, patients who received at least one dose of PD-1-mesoCAR-T and had at least one post-baseline response evaluation). Results: From July 20, 2020 to January 31, 2022, 9 patients were screened and enrolled and only 6 patients were evaluable. Mesothelin positive rate in the samples of these patients were from 50% to 100%. Three patients of them had PD-L1 positive staining in specimen. All patients underwent lymphodepletion before infusion. Two of patients (33.3%) had cytokine release syndrome, which were grade 1 and grade 3, respectively. Four patients (66.7%) developed fever. Two patients (25%) had grade 3 pulmonary infection. Stable disease was sustained for ≥3 months in 5 patients; 1 exhibited complete response and one partial response on PET or CT scan. The objective response rate (ORR) was 33.3%, but increased to 66.7% in the patients with PD-L1 positive. Only one patient of these patients appeared progression of disease after infusion. All enrolled patients are alive and observed continually. Conclusions: The clinical study of 6 patients with malignant mesothelioma shows that PD-1-mesoCAR-T cells are safe and feasible, indicating a promising curative effect. Effect of the CAR-T cells on the long term survival will be observed continually. Clinical trial information: 01722149.

Purpose: Malignant gliomas are the most common tumors in the central nervous system with a poor prognosis. Recently, CD4+ cytotoxic T cells (CTLs) are being increasingly recognized as possessing antitumor capacity. However, their presence, activity and regulation in glioma have not been investigated in detail. Methods: To examine this, 72 grade II and grade III Han Chinese glioma patients and 30 Han Chinese healthy controls were investigated. Results: We found that compared to healthy controls, glioma patients had significantly upregulated frequencies of circulating CD4+ CTLs, identified by the expression of granzyme A (GzmA), granzyme B (GzmB) and/or perforin. The stimulated CD4+ CTLs in grade II and grade III glioma patients also had less proliferative ability than those in healthy controls, a feature of suppression that progressed with tumor grade. The frequencies of GzmB-expressing circulating CD4+ CTLs were directly associated with prognosis. We hypothesized that the programed death 1 (PD-1)/PD-ligan...

Background/Aims: Gastric cancer is a highly aggressive tumor containing cancer stem cells (CSCs), which participate in tumor initiation, therapeutic resistance, and tumor relapse. Proline-rich protein 11 (PRR11) has been shown to be up-regulated in human cancers; however, its role in gastric CSCs is unknown. We hypothesize that PRR11 may affect tumorigenicity of gastric CSCs. In this study, we explored the biological function and regulation of PRR11 in gastric CSCs. Methods: Expression of PRR11 was evaluated in gastric CSC cell line by real-time quantitative PCR and western blot. The effect of PRR11 on tumorigenicity was examined by interference with gene expression using lentiviral vector-loaded shRNA. A xenograft tumor model using NOD/SCID mice was established to examine the role of PRR11 in tumor development. Results: Data showed that PRR11 was highly expressed in gastric CSCs. PRR11 was responsible for the maintenance of self-renewal and tumorigenicity of gastric CSCs, and overexpression of exogenous PRR11 could restore the self-renewal of gastric non-CSCs. Furthermore, interference with PRR11 altered the expression of stemness transcription factors. Interestingly, MAPK signaling controlled PRR11 expression by increasing PRR11 protein stability, and maintained gastric CSCs self-renewal in a PRR11 dependent manner. Conclusions: PRR11 regulated self-renewal and tumorigenicity of gastric CSCs through MAPK signaling, and could be used as a therapeutic target for gastric cancer.

Recent researches have suggested that autophagy may play critical roles in tumorigenesis. Immunity-related GTPase family M (IRGM) is a human protein highlighted for its contribution to autophagy upon inflammation and infections. Studies have shown that IRGM is involved in the development of several cancers. In the current study, we investigated expression of IRGM and gastric cancer. Levels of messenger RNA (mRNA) and protein were examined by real-time reverse transcription PCR (RT-PCR) and Western blot, respectively. Data showed that mRNA level of IRGM was significantly increased in peripheral blood mononuclear cells (PBMCs) of gastric cancer patients than in PBMCs from healthy controls (p > 0.05). Moreover, both mRNA and protein levels were significantly higher in cancer tissues compared to adjacent noncancerous stomach tissues (1.28-fold, p < 0.001; 1.19-fold, p < 0.01, respectively). However, the level of IRGM seemed not to be affected by Helicobacter pylori infection. In addition, we investigated the correlation between IRGM expression and cancer stages and identified that stage IV patients had upregulated mRNA level and protein level of IRGM in cancer tissues than those stage I patients. Our findings suggest that expression of IRGM is dysregulated in gastric cancer and that the molecule may affect progression of the disease.

Recent studies have implicated autophagy in numerous cellular responses to stress. During the establishment of human lung cancer cell lines without mitochondrial DNA, a significant depopulation of mitochondria occurred that was accompanied by the loss of the mitochondrial membrane potential. Notably, we observed autophagy in ethidium bromide (EtBr)-induced mitochondrial degradation. In the present study, we confirmed the involvement of autophagy in mitochondrial degradation after exposure to a low concentration of EtBr. Lung cancer cells undergoing mitochondrial autophagy exhibited a slower growth rate in vitro and in vivo. Furthermore, the degradation was mediated by the class III phosphatidylinositol 3-kinase (PI3K)-Beclin-1 complex. These findings indicate that autophagy is responsible for EtBr‑induced mitochondrial degradation via the PI3K‑Beclin-1 signaling pathway.

CD8+ T cells are considered to be critical in tumor surveillance and elimination. Increased CD8+ T cell frequency and function is associated with better prognosis in cancer patients. Interleukin 10 is a cytokine with controversial roles in CD8+ T cell–mediated anti-tumor immunity. We therefore examined the interleukin 10 expression and consumption in CD8+ T cells harvested from the peripheral blood and resected tumors of gastric cancer patients of stages II–IV. We found that the gastric cancer patients presented significantly elevated frequencies of interleukin 10–expressing cells in both CD4+ and CD8+ T cells compared to healthy controls. But distinctive from the interleukin 10–expressing CD4+ T cells, which increased in frequency in advanced cancer, the interleukin 10–expressing CD8+ T cells did not increase with cancer stage in the peripheral blood and actually decreased with cancer stage in resected tumor. Interleukin 10 and interleukin 10 receptor expression was also enriched in interferon gamma–expressing activated CD8+ T cells. Compared to interleukin 10–nonexpressing CD8+ T cells, interleukin 10 receptor–expressing CD8+ T cells secreted significantly elevated interferon gamma levels. Treatment of anti-CD3/CD28-stimulated, purified CD8+ T cells with interleukin 10 alone could significantly enhance CD8+ T cell survival, an effect dependent on interleukin 10 receptor expression. Interleukin 10 also increased CD8+ T cell proliferation synergistically with interferon gamma but not alone. Analysis of downstream signal transducer and activator of transcription molecules showed that interleukin 10 treatment significantly increased the phosphorylation of signal transducer and activator of transcription 3 and signal transducer and activator of transcription 1 to lesser extent. Together, these results demonstrate that interleukin 10 possessed stimulatory roles in activated CD8+ T cells from gastric cancer patients.

Overexpression of ABCG2 is considered a major mechanism of cancer drug resistance. Recent studies have shown that ABCG2 can regulate the switch between symmetric and asymmetric cell division in adult stem cells; however, the relationship between ABCG2 and cell division in drug-resistant cancer cells remains to be determined. In the present study, we demonstrated that ABCG2 is involved in the cell division of drug-resistant cancer cells. We first established drug-resistant H460 and A549 cell lines by repeated exposure to cisplatin and found that the expression of ABCG2 in these cell lines was significantly increased. As evidenced by PKH-26 staining, these drug-resistant cell lines favored symmetric division, which differed from the asymmetric division of the parental cells. Furthermore, we established stable ABCG2‑overexpressing and stable shRNA-ABCG2‑knockdown cell lines to evaluate the potential role of ABCG2 in cancer cell division. The results showed that overexpression of ABCG2 in A549 parental cells significantly increased the proportion of symmetric division, whereas knockdown of ABCG2 in drug-resistant A549 cells significantly increased the proportion of asymmetric division. Taken together, our findings suggest that ABCG2 is involved in the modulation of cancer drug resistance by regulating the pattern of cell division. The present study provides novel insight into the role of ABCG2 in cancer treatment resistance.