Engineered CAR T cells targeting mesothelin by piggyBac transposon system for the treatment of pancreatic cancer.

Patients with pancreatic cancer have a poor prognosis largely due to the poor efficacy of the available treatment modalities. In this study, we engineered mesothelin-targeting chimeric antigen receptor T cells (mesoCAR T) using the piggyBac transposon based plasmid electroporation technique for specific targeting of pancreatic cancer cells expressing mesothelin. In vitro, mesoCAR T cells exhibited rapid and robust killing effect against ASPC1 cells with high expression levels of mesothelin with high production of IFN-γ; the cytotoxic effect on PANC1 cells with low expressions of mesothelin was relatively attenuated. In the ASPC1 xenograft mice model, mesoCAR T cells significantly suppressed the tumor growth accompanied with higher-level IFN-γ secretion as compared to control T cells. Besides, more mesoCAR T cells differentiated into memory T cells after tumor remission, whilst causing minimal lesions in major organs. Our study suggests promising efficacy of piggyBac transposon-based mesoCAR T cell therapy for pancreatic cancer, which is a potential candidate for clinical translation.

Marine Lectins DlFBL and HddSBL Fused with Soluble Coxsackie-Adenovirus Receptor Facilitate Adenovirus Infection in Cancer Cells BUT Have Different Effects on Cell Survival.

Cancer development and progression are usually associated with glycosylation change, providing prognostic and diagnostic biomarkers, as well as therapeutic targets, for various cancers. In this work, Dicentrarchus labrax fucose binding lectin (DlFBL) and Haliotis discus discus sialic acid binding lectin (HddSBL) were genetically fused with soluble coxsackie-adenovirus receptor (sCAR), and produced through a bacterial expression system. Results showed that recombinant sCAR-DlFBL not only facilitated adenovirus Ad-EGFP infection in K562/ADR and U87MG cells, but also enhanced the cytotoxicity of adenovirus harboring gene encoding Pinellia pedatisecta agglutinin (PPA) or DlFBL (Ad-PPA or Ad-DlFBL) on U87MG cells through inducing apoptosis. Recombinant sCAR-HddSBL facilitated Ad-EGFP infection, but dramatically counteracted the cytotoxicity of both Ad-PPA and Ad-DlFBL in U87MG cells. Further analysis revealed that sCAR-HddSBL, but not sCAR-DlFBL, significantly upregulated transcription factor E2F1 levels in U87MG cells, which might be responsible for the adverse effect of sCAR-HddSBL on Ad-PPA and Ad-DlFBL. Taken together, our data suggested that sCAR-DlFBL could be further developed to redirect therapeutic adenoviruses to infect cancer cells such as U87MG, and the sCAR-lectin fusion proteins for adenoviral retargeting should be carefully examined for possible survival signaling induced by lectins, such as HddSBL.

Anguilla japonica lectin 1 delivery through adenovirus vector induces apoptotic cancer cell death through interaction with PRMT5.

BACKGROUND: Our previous studies have demonstrated that, through adenovirus mediated gene delivery, various exogenously expressed lectins elicited cytotoxicity to cancer cells, utilizing protein arginine methyltransferase 5 (PRMT5) as a common binding target. METHODS: In the present study, a FLAG tagged Anguilla japonica lectin 1 (AJL1) expression cassette was genetically harbored in a replication-defective adenovirus genome, forming Ad.FLAG-AJL1. The exogenous AJL1-induced cytotoxicity and the underlying mechanisms were analyzed. RESULTS: The exogenous AJL1 suppressed the proliferation of a variety of human cancer cells by inducing apoptosis. Caspase 9, Bcl-2, X-linked inhibitor of apoptosis protein, mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase were found to be responsible for the exogenous AJL1-induced cytotoxicity. AJL1 was further suggested to regulate PRMT5-E2F-1 pathway, a pathway shared by previously reported marine lectins Dicentrarchus labrax fucose binding lectin and Strongylocentrotus purpuratus rhamnose binding lectin. A localization study revealed that exogenous AJL1 widely distributed in the cell membrane and cytoplasm. CONCLUSIONS: The results of the present study suggest that the PRMT5-E2F-1 pathway may act as a common target for exogenous lectins including AJL1, and the cellular response to exogenous AJL1 may suggest a novel agent for cancer gene therapy. Copyright © 2016 John Wiley & Sons, Ltd.

Adenovirus carrying gene encoding Haliotis discus discus sialic acid binding lectin induces cancer cell apoptosis.

Lectins exist widely in marine bioresources such as bacteria, algae, invertebrate animals and fishes. Some purified marine lectins have been found to elicit cytotoxicity to cancer cells. However, there are few reports describing the cytotoxic effect of marine lectins on cancer cells through virus-mediated gene delivery. We show here that a replication-deficient adenovirus-carrying gene encoding Haliotis discus discus sialic acid binding lectin (Ad.FLAG-HddSBL) suppressed cancer cell proliferation by inducing apoptosis, as compared to the control virus Ad.FLAG. A down-regulated level of anti-apoptosis factor Bcl-2 was suggested to be responsible for the apoptosis induced by Ad.FLAG-HddSBL infection. Further subcellular localization studies revealed that HddSBL distributed in cell membrane, ER, and the nucleus, but not in mitochondria and Golgi apparatus. In contrast, a previously reported mannose-binding lectin Pinellia pedatisecta agglutinin entered the nucleus as well, but did not distribute in inner membrane systems, suggesting differed intracellular sialylation and mannosylation, which may provide different targets for lectin binding. Further cancer-specific controlling of HddSBL expression and animal studies may help to provide insights into a novel way of anti-cancer marine lectin gene therapy. Lectins may provide a reservoir of anti-cancer genes.

PiggyBac-engineered T cells expressing a glypican-3-specific chimeric antigen receptor show potent activities against hepatocellular carcinoma.

Glypican-3 (GPC3) is an attractive target for chimeric antigen receptor (CAR)-T cell therapy, as it is overexpressed in most hepatocellular carcinoma (HCC) tissues but shows restricted expression in healthy adult tissues. Herein, we generated GPC3-specific CAR-T cells for HCC therapy by electroporation with plasmid DNA encoding the piggyBac (PB) transposon and the hyperactive piggyBac transposase simultaneously instead of by commonly-used viral vectors. Our results demonstrated that GPC3CAR gene was efficiently integrated into the genome of T cells utilizing the PB transposon system. Upon stimulation with GPC3 antigen, GPC3CAR-T cells could be effectively activated, proliferate strongly and secrete high levels of cytokines. It also was demonstrated that GPC3CAR-T cells displayed potent cytotoxicity against GPC3-positive HCC cell lines in vitro by using real-time cell analyser (RTCA) system and the JuLI™ Stage Cell History Recorder. More importantly, in a Huh-7 xenograft mouse model, GPC3CAR-T cells significantly reduced the tumour burden companied with the secretion of high levels of IFN-γ. Moreover, T cells in mice treated with GPC3CAR-T cells could infiltrate into tumour tissues and persist as effector memory T cells (TEM). Overall, our study suggests that the use of PB system-based GPC3CAR-T cell therapy could be a promising clinical strategy for patients with HCC.

A SIRPα-Fc fusion protein enhances the antitumor effect of oncolytic adenovirus against ovarian cancer.

Oncolytic viruses armed with therapeutic transgenes of interest show great potential in cancer immunotherapy. Here, a novel oncolytic adenovirus carrying a signal regulatory protein-α (SIRPα)-IgG1 Fc fusion gene (termed SG635-SF) was constructed, which could block the CD47 'don't eat me' signal of cancer cells. A strong promoter sequence (CCAU) was chosen to control the expression of the SF fusion protein, and a 5/35 chimeric fiber was utilized to enhance the efficiency of infection. As a result, SG635-SF was found to specifically proliferate in hTERT-positive cancer cells and largely increased the abundance of the SF gene. The SF fusion protein was effectively detected, and CD47 was successfully blocked in SK-OV3 and HO8910 ovarian cancer cells expressing high levels of CD47. Although the ability to induce cell cycle arrest and cell death was comparable to that of the control empty SG635 oncolytic adenovirus in vitro, the antitumor effect of SG635-SF was significantly superior to that of SG635 in vivo. Furthermore, CD47 was largely blocked and macrophage infiltration distinctly increased in xenograft tissues of SK-OV3 cells but not in those of CD47-negative HepG2 cells, indicating that the enhanced antitumor effect of SG635-SF was CD47-dependent. Collectively, these findings highlight a potent antitumor effect of SG635-SF in the treatment of CD47-positive cancers.

Antitumor activity of EGFR-specific CAR T cells against non-small-cell lung cancer cells in vitro and in mice.

Effective control of non-small-cell lung cancer (NSCLC) remains clinically challenging, especially during advanced stages of the disease. This study developed an adoptive T-cell treatment through expression of a chimeric antigen receptor (CAR) to target human epidermal growth factor receptor (EGFR) in NSCLC. We optimized the non-viral piggyBac transposon system to engineer human T cells for the expression of EGFR-CAR, consisting of EGFR scFv, transmembrane domain, and intracellular 4-1BB-CD3ζ signaling domains. The modified CAR T cells exhibited expansion capability and anticancer efficacy in a time- and antigen-dependent manner in vitro as well as regression of EGFR-positive human lung cancer xenografts in vivo. EGFR-CAR T therapy is a promising strategy to improve the efficacy and potency of the adoptive immunotherapy in NSCLC. Moreover, EGFR-CAR T therapy could become a clinical application for NSCLC patients in the future.

CD47-retargeted oncolytic adenovirus armed with melanoma differentiation-associated gene-7/interleukin-24 suppresses in vivo leukemia cell growth.

Our previous studies have suggested that harboring a soluble coxsackie-adenovirus receptor-ligand (sCAR-ligand) fusion protein expression cassette in the viral genome may provide a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resisting to serotype 5 adenovirus infection. We report here a novel oncolytic adenovirus vector redirected to CD47+ leukemia cells though carrying a sCAR-4N1 expression cassette in the viral genome, forming Ad.4N1, in which 4N1 represents the C-terminal CD47-binding domain of thrombospondin-1. The infection and cytotoxicity of Ad.4N1 in leukemia cells were determined to be mediated by the 4N1-CD47 interaction. Ad.4N1 was further engineered to harbor a gene encoding melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), forming Ad.4N1-IL24, which replicated dramatically faster than Ad.4N1, and elicited significantly enhanced antileukemia effect in vitro and in a HL60/Luc xenograft mouse model. Our data suggest that Ad.4N1 could act as a novel oncolytic adenovirus vector for CD47+ leukemia targeting gene transfer, and Ad.4N1 harboring anticancer genes may provide novel antileukemia agents.

Exogenous expression of marine lectins DlFBL and SpRBL induces cancer cell apoptosis possibly through PRMT5-E2F-1 pathway.

Lectins are widely existed in marine bioresources, and some purified marine lectins were found toxic to cancer cells. In this report, genes encoding Dicentrarchus labrax fucose-binding lectin (DlFBL) and Strongylocentrotus purpuratus rhamnose-binding lectin (SpRBL) were inserted into an adenovirus vector to form Ad.FLAG-DlFBL and Ad.FLAG-SpRBL, which elicited significant in vitro suppressive effect on a variety of cancer cells. Anti-apoptosis factors Bcl-2 and XIAP were determined to be downregulated by Ad.FLAG-DlFBL and Ad.FLAG-SpRBL. Subcellular localization studies showed that DlFBL but not SpRBL widely distributed in membrane systems. Both DlFBL and SpRBL were shown associated with protein arginine methyltransferase 5 (PRMT5), and PRMT5-E2F-1 pathway was suggested to be responsible for the DlFBL and SpRBL induced apoptosis. Further investigations revealed that PRMT5 acted as a common binding target for various exogenous lectin and non-lectin proteins, suggesting a role of PRMT5 as a barrier for foreign gene invasion. The cellular response to exogenous lectins may provide insights into a novel way for cancer gene therapy.