Binghe Tan

Abstract Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies 1–7 . However, CAR-T cell therapy currently has several limitations 8–12 . Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR–Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR + cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1 -integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.

Abstract Therapies targeting immune checkpoints have shown great clinical potential in a subset of patients with cancer but may be hampered by a failure to reverse the immunosuppressive tumor microenvironment (TME). As the most abundant immune cells in TME, tumor-associated macrophages (TAM) play nonredundant roles in restricting antitumor immunity. The leucine-rich repeat-containing G-protein–coupled receptor 4 (Lgr4, also known as Gpr48) has been associated with multiple physiologic and pathologic functions. Lgr4 and its ligands R-spondin 1–4 have been shown to promote the growth and metastasis of tumor cells. However, whether Lgr4 can promote tumor progression by regulating the function of immune cells in the tumor microenvironment remains largely unknown. Here, we demonstrate that Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Notably, urethane-induced lung carcinogenesis, Lewis lung carcinoma (LLC), and B16F10 melanoma tumors were all markedly reduced in Lgr4fl/flLyz2cre/+ mice, characterized by fewer protumoral M2 TAMs and increased CD8+ T lymphocyte infiltration in the TME. Furthermore, LLC tumor growth was greatly depressed when Rspo/Lgr4/Erk/Stat3 signaling was blocked with either the LGR4 extracellular domain or an anti-Rspo1 antibody. Importantly, blocking Rspo-Lgr4 signaling overcame LLC resistance to anti-PD-1 therapy and improved the efficacy of PD-1 immunotherapy against B16F10 melanoma, indicating vital roles of Rspo-Lgr4 in host antitumor immunity and a potential therapeutic target in cancer immunotherapy. Significance: This study identifies a novel receptor as a critical switch in TAM polarization whose inhibition sensitizes checkpoint therapy–resistant lung cancer to anti-PD-1 therapy. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4929/F1.large.jpg. Cancer Res; 78(17); 4929–42. ©2018 AACR.

Abstract P2X7, a crucial sensor of extracellular ATP, is widely distributed in different immune cells as a potent stimulant of inflammation and immunity. P2X7 is also highly expressed in immunosuppressive cells such as tumor-associated macrophages (TAM) and even tumor cells. However, the function and potential applications of P2X7-mediated immunosuppressive responses in the tumor microenvironment remain unclear. Here, we demonstrated that P2X7 was highly expressed in TAMs and that P2X7 deficiency impaired the “M2-like” polarization of TAMs via downregulation of STAT6 and IRF4 phosphorylation both in vivo and in vitro. P2X7 deficiency restricted the progression of urethane-induced lung carcinogenesis and Lewis lung cancer by decreasing tumor cell proliferation and angiogenesis, promoting T-cell mobilization, and reversing M2-like TAM polarization. Thus, deletion or blockade of P2X7 was therapeutic for lung cancer. Furthermore, resistance to both immunotherapy (anti–PD-1 antibody) and chemotherapy (cisplatin) was overcome by coadministration of the P2X7 inhibitors O-ATP, A-438079 hydrochloride, and A-740003. Therefore, our data revealed a vital role of P2X7 in tumor formation through regulating TAM polarization, suggesting the therapeutic potential of P2X7 blockade in patients with lung cancer.

Extracellular nucleotides that constitute a "danger signal" play an important role in the regulation of immune responses. However, the function and mechanism of extracellular UDP and P2Y6 in antiviral immunity remain unknown. In this study, we demonstrated the in vitro and in vivo protection of UDP/P2Y6 signaling in vesicular stomatitis virus (VSV) infection. First, we demonstrated that VSV-infected cells secrete UDP from the cytoplasm as a danger signal to arouse surrounding cells. Meanwhile, expression of the UDP-specific receptor P2Y6 also was enhanced by VSV. Consequently, UDP protects RAW 264.7 cells, murine embryonic fibroblasts, bone marrow-derived macrophages, and L929 cells from VSV and GFP lentivirus infection. This protection can be blocked by the P2Y6 selective antagonist MRS2578 or IFN-α/β receptor-blocking Ab. VSV-induced cell death and virus replication were both enhanced significantly by knocking down and knocking out P2Y6 in different cells. Mechanistically, UDP facilitates IFN-β secretion through the p38/JNK- and ATF-2/c-Jun-signaling pathways, which are crucial in promoting antiviral immunity. Interestingly, UDP was released through a caspase-cleaved pannexin-1 channel in VSV-induced apoptotic cells and protected cells from infection through P2Y6 receptor in an autocrine or paracrine manner. Furthermore, UDP also protected mice from VSV infection through P2Y6 receptors in an acute neurotropic infection mouse model. Taken together, these results demonstrate the important role of extracellular UDP and P2Y6 as a danger signal in antiviral immune responses and suggest a potential therapeutic role for UDP/P2Y6 in preventing and controlling viral diseases.