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Conditionally replicating adenoviruses (CRAd) have been under extensive investigations as anticancer agents. Previously, we found that ZD55, an adenovirus serotype 5-based CRAd, infected and killed the leukemia cells expressing coxsackie adenovirus receptor (CAR). However, majority of leukemic cells lack CAR expression on their cell surface, resulting in resistance to CRAd infection. In this study, we showed that SG235, a novel fiber chimeric CRAd that has Ad35 tropism, permitted CAR-independent cell entry, and this in turn produced selective cytopathic effects in a variety of human leukemic cells in vitro and in vivo. Moreover, SG235 expressing exogenous tumor necrosis factor-related apoptosis-inducing ligand (SG235-TRAIL) effectively induced apoptosis of leukemic cells via the activation of extrinsic and intrinsic apoptotic pathway and elicited a superior antileukemia activity compared with SG235. In addition, normal hematopoietic progenitors were resistant to the inhibitory activity of SG235 and SG235-TRAIL. Our data suggest that these novel oncolytic agents may serve as useful tools for the treatment of leukemia. [Mol Cancer Ther 2009;8(5):1387–97]

Supplementary Fig. S1 from Effective gene-viral therapy of leukemia by a new fiber chimeric oncolytic adenovirus expressing TRAIL: in vitro and in vivo evaluation

Recently, mesenchymal stem cell (MSC) therapy has been suggested as an effective alternative approach for the treatment of hepatic diseases. MSCs have potential therapeutic value, because they have high self-renewal ability, are capable of multipotent differentiation, and have low immunogenicity. Furthermore, MSCs have the potential to differentiate into hepatocytes, and the therapeutic value exists in their immune-modulatory properties and secretion of trophic factors, such as growth factors and cytokines. Moreover, MSCs can suppress inflammatory responses, reduce hepatocyte apoptosis, increase hepatocyte regeneration, regress liver fibrosis, and enhance liver functionality.

CD19-directed chimeric antigen receptor (CAR) T cell therapy has been shown to achieve a considerably durable response in patients with refractory or relapsed B cell non-Hodgkin lymphomas, as seen from the results of Zuma-1, Zuma-5, and other clinical trials. Most of these CARs were generated by lentivirus or reverse adenovirus. It is rare to see CARs using non-viral vectors, such as Piggy Bac (pb), in treating lymphoma patients with active diseases. Generally, patients with a high tumor burden tend to have a higher rate of severe cytokine release syndrome (CRS) or neurological events as reported in the literature. Patients with symptomatic pleural effusions are excluded from the Zuma-1 trial because of the risk of severe CRS. We report here that a patient with relapsed follicular lymphoma with bulky disease and massive chylous ascites failed several lines of chemotherapy. After infusion of the CD19-directed pbCAR-T cells at 6 × 106 cells/kg, the patient had a rapid response and achieved a nearly complete metabolic remission on day 28. There was only grade 1 CRS, and no neurotoxicity occurred. The CAR-T cells reached a peak level on day 14 and spread into the ascites and expanded for 3 months. This might be the first case reported for pbCAR-T cells to treat relapsed follicular lymphoma directly. The long-term efficacy will be observed, and more patients be tested in the future.Clinical Trial Registrationhttps://ClinicalTrials.gov, identifier NCT05472610.

Both ZeBrafish (

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.

Immunotherapies, such as monoclonal antibody therapy and checkpoint inhibitor therapy, have shown inspiring clinical effects for the treatment of cancer. Chimeric antigen receptor T (CAR-T) cells therapy was an efficacious therapeutic approach treating hematological malignancies and encouraging results have been achieved. Three kinds of CAR-T cell therapies, Kymriah (tisagenlecleucel), Yescarta (axicabtagene ciloleucel), were approved for clinical application in 2017 and Tecartus (brexucabtagene autoleucel) was approved in 2020. Despite some progress have been made in treating multiple hematologic tumors, threats still remain for the application of CAR-T cell therapy considering its toxicities and gaps in knowledge. To further comprehend present research status and trends, the review concentrates on CAR-T technologies, applications, adverse effects and safety measures about CAR-T cell therapy in hematological neoplasms. We believe that CAR-T cell therapy will exhibit superior safety and efficacy in the future and have potential to be a mainstream therapeutic choice for the elimination of hematologic tumor.

Objective The evaluation of lymphocyte subsets is widely regarded as an important factor for monitoring tumor progression and response to therapy. This study was designed to establish a comprehensive and detailed assessment of peripheral lymphocyte subsets with a multi-parametric flow cytometry assay for response prediction and prognosis evaluation of cancer patients. Methods Peripheral blood samples collected from 40 cancer patients and 23 age- and sex-matched healthy volunteers were tested for 29 lymphocyte subsets by flow cytometry. The univariate analysis was applied to establish the reference interval of healthy samples, and the ratio and proportion of 29 lymphocyte subsets between patient samples and healthy controls were compared to evaluate their clinical significance by Mann-Whitney U-test model. Results The reference ranges of 29 lymphocyte subsets were established with a normal distribution and no significant differences were observed between genders. Compared with healthy control group, lower proportion and ratio of specific parameters, such as Naive Th cells (p

Nonviral transposon piggyBac (PB) and lentiviral (LV) vectors have been used to deliver chimeric antigen receptor (CAR) to T cells. To understand the differences in the effects of PB and LV on CAR T-cell functions, a CAR targeting CD19 was cloned into PB and LV vectors, and the resulting pbCAR and lvCAR were delivered to T cells to generate CD19pbCAR and CD19lvCAR T cells. Both CD19CAR T-cell types were strongly cytotoxic and secreted high IFN-γ levels when incubated with Raji cells. TNF-α increased in CD19pbCAR T cells, whereas IL-10 increased in CD19lvCAR T cells. CD19pbCAR and CD19lvCAR T cells showed similar strong anti-tumor activity in Raji cell-induced mouse models, slightly reducing mouse weight while enhancing mouse survival. High, but not low or moderate, concentrations of CD19pbCAR T cells significantly inhibited Raji cell-induced tumor growth in vivo. These CD19pbCAR T cells were distributed mostly in mesenteric lymph nodes, bone marrow of the femur, spleen, kidneys, and lungs, specifically accumulating at CD19-rich sites and CD19-positive tumors, with CAR copy number being increased on day 7. These results indicate that pbCAR has its specific activities and functions in pbCAR T cells, making it a valuable tool for CAR T-cell immunotherapy.

“On-target off-tumor” toxicity is a major challenge to the use of chimeric antigen receptor (CAR)-engineered T cells in the treatment of solid malignancies, because of the expression of target antigens in normal tissues. Mesothelin overexpression is associated with poor prognosis of multiple solid tumors, and would therefore appear to be a suitable antigen target. To understand the risk of toxicity to different organs on anti-mesothelin CAR T cell therapy, single-cell RNA sequencing (scRNA-seq) datasets derived from major human physiological systems were analyzed in this study, including the respiratory, cardiovascular, digestive, and urinary systems. According to scRNA-seq datasets, the organs were stratified into high or low risk based on the level of mesothelin expression. We report that the proportion of mesothelin-positive cells was 7.71%, 2.40% and 2.20% of myocardial cells, pulmonary cells and stomach cells, respectively, indicating that these organs could be at high risk of “on-target off-tumor” toxicity on anti-mesothelin CAR T cell therapy. By contrast, esophagus, ileum, liver, kidney and bladder exhibited low mesothelin expression (

Both ZeBrafish (ZB), a recently identified DNA transposon in the zebrafish genome, and SB, a reconstructed transposon originally discovered in several fish species, are known to exhibit high transposition activity in vertebrate cells. Although a similar structural organization was observed for ZB and SB transposons, the evolutionary profiles of their homologs in various species remain unknown. In the present study, we compared their taxonomic ranges, structural arrangements, sequence identities, evolution dynamics, and horizontal transfer occurrences in vertebrates. In total, 629 ZB and 366 SB homologs were obtained and classified into four distinct clades, named ZB, ZB-like, SB, and SB-like. They displayed narrow taxonomic distributions in eukaryotes, and were mostly found in vertebrates, Actinopterygii in particular tended to be the major reservoir hosts of these transposons. Similar structural features and high sequence identities were observed for transposons and transposase, notably homologous to the SB and ZB elements. The genomic sequences that flank the ZB and SB transposons in the genomes revealed highly conserved integration profiles with strong preferential integration into AT repeats. Both SB and ZB transposons experienced horizontal transfer (HT) events, which were most common in Actinopterygii. Our current study helps to increase our understanding of the evolutionary properties and histories of SB and ZB transposon families in animals.

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.

This phase I clinical trial is designed to assess the safety and feasibility of the epidermal growth factor receptor (EGFR) chimeric antigen receptor (CAR) T-cell generated by the piggyBac transposon system in advanced relapsed/refractory non-small cell lung cancer (NSCLC) patients. Compared to viral systems, the piggyBac transposon system is a simpler, more economical, and alternative way to introduce chimeric antigen receptor (CAR) transgenes into T cells. This study recruited nine patients with advanced relapsed/refractory EGFR-positive NSCLC for two cycles of the piggyBac-generated EGFR-CAR T cells at dose of 1 × 106 cells/kg or 3 × 106 cells/kg of body weight. The patients were monitored for adverse events, clinical response, and persistence of plasma GFR-CAR T cells. Infusions of piggyBac-generated EGFR-CAR T cells were well tolerated in all nine patients. The most common adverse events were grade 1 to 3 fever and there were no patients who experienced grade 4 adverse events or serious cytokine release syndrome. After treatment, eight of nine patients showed detectable EGFR-CAR T cells in their peripheral blood. One patient showed a partial response and lasted for more than 13 months, while six had stable disease, and two had progressed disease. The progression-free survival of these nine patients was 7.13 months (95% CI 2.71–17.10 months), while the median overall survival was 15.63 months (95% CI 8.82–22.03 months). This Phase I clinical trial revealed that the non-viral piggyBac transposon system-engineered EGFR-CAR T-cell therapy is feasible and safe in treatment of EGFR-positive advanced relapsed/refractory NSCLC patients. Future study will assess it in more patients or even possibly with a higher dose. Trial registration number NCT03182816.

Background Although chimeric antigen receptor (CAR)-T cell therapy has been remarkably successful for haematological malignancies, its efficacy against solid tumors is limited. The combination of CAR-T cell therapy with immune checkpoint inhibitors (CPIs), such as PD-1, PD-L1, and CTLA-4 antibodies, is a promising strategy for enhancing the antitumor efficacy of CAR-T cells. However, because most patients acquire resistance to CPIs, investigating other strategies is necessary to further improve the antitumor efficacy of CAR-T cell therapy for solid tumors. Recently, CD40 agonist antibodies showed potential antitumor efficacy by activating the CD40 pathway. Results Based on the piggyBac transposon system, rather than the widely used viral vectors, we constructed a meso3-CD40 CAR-T targeting region III of mesothelin (MSLN) that possessed the ability to secrete anti-CD40 antibodies. Compared with meso3 CAR-T cells, which did not secrete the anti-CD40 antibody, meso3-CD40 CAR-T cells secreted more cytokines and had a relatively higher proportion of central memory T (TCM) cells after stimulation by the target antigen. In addition, compared with meso3 CAR-T cells, meso3-CD40 CAR-T cells had a more powerful cytotoxic effect on target cells at a relatively low effector-to-target ratio. More importantly, we demonstrated that the antitumor activity of meso3-CD40 CAR-T cells was enhanced in a human ovarian cancer xenograft model in vivo. Conclusions In conclusion, these results highlight anti-CD40-secreting CAR-T cells generated by nonviral vectors as a potential clinical strategy for improving the efficacy of CAR-T cell therapies.

T cells modified to co-express cytokine or other factors with chimeric antigen receptor (CAR) can induce substantial and persistent increases in antitumor capacity in vivo. However, the uncontrolled expression of cytokines or factors can lead to the overactivation of immune cells, causing severe adverse events such as cytokine release syndrome (CRS) and neurotoxicity by CAR T cells with excessive growth potential. Conventional promoters are unregulated, and their expression is unlimited in T cells. In this study, by connecting the cytomegalovirus (CMV) enhancer, core interferon gamma (IFN-γ) promoter, and a T-lymphotropic virus long terminal repeat sequence (TLTR), we constructed and screened the chimeric promoter CIFT, which was highly expressed in some cell lines secreting IFN-γ and silenced in others. We placed this promoter upstream of the anti-programmed cell death protein 1 (anti-PD-1) antibody gene, and this construct was co-transfected with the CAR construct into T cells. In vitro or in vivo, CAR T cells showed increased secretion of anti-PD-1 antibody under control of the chimeric promoter CIFT. pS-CIFT-αPD-1/CAR T also had similar or lower PD-1 expression, higher levels of T cell activation, more release of IFN-γ, and better antitumor activity specifically against mesothelin-positive and PD-1 ligand 1 (PD-L1)-positive cell lines. The chimeric promoter may be a promising strategy to manipulate the content of immune checkpoint inhibitors or other proteins in future therapeutic approaches for cancer treatment., Graphical Abstract, Chimeric antigen receptor (CAR) T cell excessive growth always leads to severe adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity. The source of this issue is considered to be the unbridled expression of cytokines or immune checkpoint inhibitors, which is caused by ineligible chimeric promoters. In this study, Qian and colleagues developed a chimeric promoter that is able to regulate anti-PD-1 antibody expression in CAR T cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

As non-viral transgenic vectors, the piggyBac transposon system represents an attractive tool for gene delivery to achieve a long-term gene expression in immunotherapy applications due to its large cargo capacity, its lack of a trace of transposon and of genotoxic potential, and its highly engineered structure. However, further improvements in transpose activity are required for industrialization and clinical applications. Herein, we established a one-plasmid effective screening system and a two-step high-throughput screening process in yeast to isolate hyperactive mutants for mammalian cell applications. By applying this screening system, 15 hyperactive piggyBac transposases that exhibited higher transpose activity compared with optimized hyPBase in yeast and four mutants that showed higher transpose activity in mammalian cells were selected among 3000 hyPBase mutants. The most hyperactive transposase, bz-hyPBase, with four mutation sites showed an ability to yield high-efficiency editing in Chinese hamster ovarian carcinoma (CHO) cells and T cells, indicating that they could be expanded for gene therapy approaches. Finally, we tested the potential of this screening system in other versions of piggyBac transposase.

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.

Background: Infiltration of immune cells and immune microenvironment determine the proliferative activity of the tumor and metastasis. The aim of this study was to analyze the influence of activation or suppression of the immune response mediators on the prognosis of biliary tract cancer (BTC). Methods: We searched Pubmed, Web of Science, Embase and The Cochrane Library for relevant literatures until June 2016. The quality of studies was assessed by QUADAS-2 and NOS tools. Forest and funnel plots and all statistical analyses were generated by using Review Manager 5.3. The bias of included studies was estimated by Egger's test using Meta R package. Results: A total of 2339 patients from 12 studies were finally enrolled in this meta-analysis. Patients with high expression of immune active factors, intraepithelial tumor-infiltrating CD4+ , CD8+, and Foxp3+ T lymphocytes, MHC I, NKG2D, showed a better overall survival (OS) than those with low expression (HR=0.52, 95% CI=0.41-0.67, P

Objective The evaluation of lymphocyte subsets is widely regarded as an important factor for monitoring tumor progression and response to therapy. This study was designed to establish a comprehensive and detailed assessment of peripheral lymphocyte subsets with a multi-parametric flow cytometry assay for response prediction and prognosis evaluation of cancer patients. Methods Peripheral blood samples collected from 40 cancer patients and 23 age- and sex-matched healthy volunteers were tested for 29 lymphocyte subsets by flow cytometry. The univariate analysis was applied to establish the reference interval of healthy samples, and the ratio and proportion of 29 lymphocyte subsets between patient samples and healthy controls were compared to evaluate their clinical significance by Mann–Whitney U-test model. Results The reference ranges of 29 lymphocyte subsets were established with a normal distribution and no significant differences were observed between genders. Compared with healthy control group, lower proportion and ratio of specific parameters, such as Naïve Th cells (p

V-set and transmembrane domain-containing 1 (VSTM1), which is downregulated in bone marrow cells from leukemia patients, may provide a diagnostic and treatment target. Here, a triple-regulated oncolytic adenovirus was constructed to carry a VSTM1 gene expression cassette, SG611-VSTM1, and contained the E1a gene with a 24-nucleotide deletion within the CR2 region under control of the human telomerase reverse transcriptase promoter, E1b gene directed by the hypoxia response element, and VSTM1 gene controlled by the cytomegalovirus promoter. Real-time quantitative PCR and Western blot analyses showed that SG611-VSTM1 expressed VSTM1 highly efficiently in the human leukemic cell line K562 compared with SG611. In Cell Counting Kit-8 and flow cytometric assays, SG611-VSTM1 exhibited more potent anti-proliferative and pro-apoptotic effects in leukemic cells compared with SG611 and exerted synergistic cytotoxicity with low-dose daunorubicin (DNR) in vitro. In xenograft models, SG611-VSTM1 intratumorally injected at a dose of 1 × 10(9) plaque forming units combined with intraperitoneally injected low-dose DNR displayed significantly stronger antitumor effects than either treatment alone. Histopathologic examination revealed that SG611-VSTM1 induced apoptosis of leukemic cells. These results implicate an important role for VSTM1 in the pathogenesis of leukemia, and SG611-VSTM1 may be a promising agent for enhancing chemosensitivity in leukemia therapy.

3039 Background: The limitations of chimeric antigen receptor T cells (CAR-T) in solid tumors are immunosuppressive tumor microenvironment and difficult infiltration to tumor. In order to reduce on-target off-tumor toxicities and circumvent the immune-suppressive tumor microenvironment(TME), we modified autologous CAR-T to be specific for mesothelin (MSLN) on cancer cells and secrete PD-1 antibodies (aPD1-MSLN-CAR T cells). Here, we report the safety and efficacy of aPD1-MSLN-CAR T cells in 10 patients with relapsed/refractory solid cancers in this single-arm, open-label, first-in-human phase I pilot study (ClinicalTrial.gov: NCT03615313). Methods: aPD1-MSLN-CAR T cells were prepared from peripheral blood mononuclear cells and engineered using PiggyBac Transposon System to target MSLN and secrete PD-1 antibodies. 10 patients with mesothelin positive relapsed/refractory solid cancers after failure to standard therapies were treated with aPD1-MSLN-CAR T cells for two or more cycles until disease progression or intolerable toxicity. The dose escalation of CAR T cells was designed to be 5×106/kg, 5×107/kg, and 1×108/kg, respectively. Adverse events were evaluated according to CTCAE-V4.03 and clinical response was assessed by RECIST 1.1. CAR expression was analyzed using quantitative real-time polymerase chain reaction. PD-1 antibodies were detected by ELISA. Serum IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α were measured using flow cytometry. Results: The most common adverse events were mild fatigue and fever. Abdominal pain was also observed in 1 patient. Grade 1 and 2 cytokine release syndromes were observed without neurologic symptoms in 10 patients. After aPD1-MSLN-mRNA-CAR T cells treatment, 2 patients (20%) achieved partial response (PR), 4 (40%) remained stable (SD), and the rest 4 (40%) patients developed disease progression (PD). The median PFS was 97 days [95% CI (13, 180)] estimated by Kaplan-Meier method. Conclusions: These findings lend support that the combination of modified CAR T cells targeting MSLN with PD1 inhibition for solid tumors is safe. Modified CAR-T cells expressing PD-1 antibodies maybe an option to improve CAR-T efficacy as a result of refined TME. Clinical trial information: NCT03615313 . [Table: see text]

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.

Immunotherapy has become the fourth cancer therapy after surgery, chemotherapy, and radiotherapy. In particular, immune checkpoint inhibitors are proved to be unprecedentedly in increasing the overall survival rates of patients with refractory cancers, such as advanced melanoma, non-small cell lung cancer, and renal cell carcinoma. However, inhibitor therapies are only effective in a small proportion of patients with problems, such as side effects and high costs. Therefore, doctors urgently need reliable predictive biomarkers for checkpoint inhibitor therapies to choose the optimal therapies. Here, we review the biomarkers that can serve as potential predictors of the outcomes of immune checkpoint inhibitor treatment, including tumor-specific profiles and tumor microenvironment evaluation and other factors.

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.

Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis. Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis. In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors. A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.

Hepatocellular carcinoma (HCC) is a major primary liver malignancy in adults. Despite the progress made, the outcome of the treatment to this disease is less than satisfactory as the post therapy tumor recurrence is almost inevitable. Accumulating pieces of evidence have suggested that the recurrence is due to the existence of a subpopulation of the HCC cells that possess the properties of stem cells and are resistant to radiation and chemotherapy. It is therefore important to understand the characteristics of this subpopulation of HCC cells, and which requires the establishment of an in vitro system to study these stem-like cancer cells. However, despite extensive efforts, the progress in establishing such an in vitro system has been slow largely due to the lack of definitive biomarkers in the isolation and expansion of these cells. In order to successfully maintain and expand HCC CSCs, we first optimized the culture system. We establish a novel medium system that allows the culture and enrichment of these hepatic stem-like cancer cells from both hepatoma cells and human primary HCC cells. These cells exhibited typical stem cell properties, such as enhanced stem cell markers, gain of EMT properties and drug resistance, and more importantly, stronger tumor-initiating capabilities. The medium may help to establish an in vitro model for hepatic cancer stem cell (HCSC) studies, which may contribute to the development of novel cell therapies and new drugs for the treatment of HCC.

AGO2 (Argonaute 2, EIF2C2) is the only member in AGO family with catalytic activity and of extreme importance during small RNAs guided gene silencing processes. The structural investigations have provided insights into details and functional mechanisms of the four major domains within AGO2. As a multifunction player, AGO2 has been revealed involved in tumorgenesis through miRNAs-dependent or independent ways. And nowadays, AGO2 has also been more importantly found ectopically over-expressed in carcinomas and closely associated with aspects of cancers in means of interacting with well-known tumor factors. Here, we provide a review on structural insights, functional mechanisms, novel roles and relationship with carcinomas of AGO2.

Adeno-associated virus (AAV) is a promising viral vector and meets most requirements of being a safe biological agent. However, the commercialization of AAV has been hampered due to the limitation of large-scale production, and only a small number of clinical trials have been launched. In recent years, progresses in scalable manufacturing of AAV have dramatically improved AAV-based clinical researches, and have assisted the development of investigational drug products. An AAV1-based investigational product, Glybera, has been formally approved by European Commission for the treatment of lipoprotein lipase deficiency (LPLD). Glybera was the first gene therapy product in the western world, and the production process involves a scalable baculovirus-insect cell system. However, many problems still need to be solved to improve the productivity and quality of AAV. The present review gives critical insights into current state-of-the-art scalable producing methodologies of AAV, such as baculovirus-insect cell system, HSV complementation system, and Ad complementation system, along with a discussion on the problems, solutions, and developmental trends. Novel AAV-producing platforms in Saccharomyces cerevisiae and vaccinia virus complementation system will also be discussed.

Cirrhosis is the long‑term outcome of chronic hepatic injury and no effective therapy is currently available for this disease. Mesenchymal stromal cells (MSCs) are multipotent cells that are easily acquired and amplified, and may be potential candidates for cell therapy against cirrhosis. This study aimed to determine the therapeutic effects of human umbilical cord‑derived MSCs (hUCMSCs) for the treatment of liver cirrhosis and identify an effective method for engrafting MSCs. The model of liver cirrhosis was established by induction of diethylnitrosamine (DEN) in rats. The isolated hUCMSCs were identified by morphology, flow cytometry and multilineage differentiation; they were injected into the vein of DEN‑induced rats at varied cell doses and infusion times. Biochemical analyses of the serum and histopathological analysis of the liver tissues were performed to evaluate the therapeutic effects of hUCMSCs in all treatment groups. The results indicated that isolated hUCMSCs were capable of self‑replication and differentiated into multiple lineages, including osteoblast‑, adipocyte‑ and hepatocyte‑like cells. Compared with the control group, administration of hUCMSCs at different cell doses and infusion times relieved DEN‑induced cirrhosis to varying degrees. The therapeutic effects of hUCMSCs on liver cirrhosis gradually improved with increased cell dose and infusion times. The improvement of cirrhosis was due to the capacity of hUCMSCs to breakdown collagen fibers in the liver. It was demonstrated that infusion of hUCMSCs effectively relieved liver cirrhosis by facilitating the breakdown of collagen fibers in a dose‑dependent manner and multiple infusions caused a relatively greater improvement in cirrhosis compared with a single infusion of hUCMSCs.

// Yin Tong 1,* , Liangshun You 1,* , Hui Liu 1 , Lu Li 1 , Haitao Meng 1 , Qijun Qian 2 , Wenbin Qian 1* 1. Institute of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University, P.R. China 2. Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, P.R. China * Contributed equally to this work Correspondence: Wenbin Qian, email: // Keywords : leukemia; oncolytic adenovirus; autophagy; Beclin-1; autophagic cell death; UVRAG Received : May 15, 2013, Accepted : May 31, 2013, Published : June 3, 2013 Abstract An attractive strategy among adenovirus-based oncolytic systems is to design adenoviral vectors to express pro-apoptotic genes, in which this gene-virotherapy approach significantly enhances tumor cell death by activating apoptotic pathways. However, the existence of cancer cells with apoptotic defects is one of the major obstacles in gene-virotherapy. Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia. A Beclin-1 cDNA was cloned in an oncolytic adenovirus with chimeric Ad5/11 fiber (SG511-BECN). SG511-BECN treatment induced significant autophagic cell death, and resulted in enhanced cell killing in a variety of leukemic cell lines and primary leukemic blasts. SG511-BECN effects were seen in chronic myeloid leukemia and acute myeloid leukemia with resistance to imatinib or chemotherapy, but exhibited much less cytotoxicity on normal cells. The SG511-BECN-induced autophagic cell death could be partially reversed by RNA interference knockdown of UVRAG, ATG5, and ATG7. We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro. In murine leukemia models, SG511-BECN prolonged the survival and decreased the xenograft tumor size by inducing autophagic cell death. Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis.

Rat embryonic stem (ES) cells hold great interest for the research of neurodevelopment and neurodegenerative diseases. However, neural conversion of rat ES cells in vitro has proven to be a challenge owing to the proliferation arrest and apoptosis. Here we report that rat ES cells can commit efficiently to a neural fate in the presence of CHIR99021 and Y-27632 (CY medium). In addition, CHIR99021 is crucial for maintaining the metabolic activity of differentiated rat ES cells, while Y-27632 facilitates the neural differentiation of rat ES cells by inhibiting bone morphogenetic protein expression. The chemical-defined CY medium also provides a platform for exploring the mechanism of neural commitment and optimizing the production efficiency of neural precursor from rat ES cells.

Purpose Cellular immunotherapy has appeared to be a promising modality for the treatment of malignant tumor. The objective of this study was to evaluate the efficacy of cellular immunotherapy combined with minimally invasive therapy. Methods We searched PubMed, Web of Science and The Cochrane Library through March 2016 for relevant studies. Short-term efficacy (the disease control rate, the control rate of quality life and the AFP descent rate) and long-term efficacy (overall survival (OS) and progression-free survival (PFS) rate) were compared as the major outcome measures. The meta-analysis was performed using Review Manager 5.3. Results A total of 1174 references in 3 databases were found of which 19 individual studies with 1774 HCC patients enrolled in this meta-analysis. Meta-analysis results showed that cellular immunotherapy combined with minimally-invasive treatment significantly improved the measures of short-term response (the disease control rate (OR = 5.91, P = 0.007), the control rate of quality lift (OR = 3.38, P = 0.003) and the AFP descent rate (OR = 4.48, P = 0.02)). Also higher 6-month PFS (OR = 2.78, P = 0.05), ≥12-month PFS (OR = 3.56, P

Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells.

Cancer is one of the major leading death causes of diseases. Prevention and treatment of cancer is an important way to decrease the incidence of tumorigenesis and prolong patients' lives. Subversive achievements on cancer immunotherapy have recently been paid much attention after many failures in basic and clinical researches. Based on deep analysis of genomics and proteomics of tumor antigens, a variety of cancer vaccines targeting tumor antigens have been tested in preclinical and human clinical trials. Many therapeutic cancer vaccines alone or combination with other conventional treatments for cancer obtained spectacular efficacy, indicating the tremendously potential application in clinic. With the illustration of underlying mechanisms of cancer immune regulation, valid, controllable, and persistent cancer vaccines will play important roles in cancer treatment, survival extension and relapse and cancer prevention. This chapter mainly summarizes the recent progresses and developments on cancer vaccine research and clinical application, thus exploring the existing obstacles in cancer vaccine research and promoting the efficacy of cancer vaccine.

Cancer is one of the major leading death causes of diseases. Prevention and treatment of cancer is an important way to decrease the incidence of tumorigenesis and prolong patients' lives. Subversive achievements on cancer immunotherapy have recently been paid much attention after many failures in basic and clinical researches. Based on deep analysis of genomics and proteomics of tumor antigens, a variety of cancer vaccines targeting tumor antigens have been tested in preclinical and human clinical trials. Many therapeutic cancer vaccines alone or combination with other conventional treatments for cancer obtained spectacular efficacy, indicating the tremendously potential application in clinic. With the illustration of underlying mechanisms of cancer immune regulation, valid, controllable, and persistent cancer vaccines will play important roles in cancer treatment, survival extension and relapse and cancer prevention. This chapter mainly summarizes the recent progresses and developments on cancer vaccine research and clinical application, thus exploring the existing obstacles in cancer vaccine research and promoting the efficacy of cancer vaccine.

Aberrant JAK/STAT3 pathway has been reported to be related to hepatocellular carcinoma (HCC) in many cell lines. In this study, a double-regulated oncolytic adenovirus vector that can replicate and induce a cytopathic effect in alpha-fetoprotein (AFP)-positive HCC cell lines with p53 dysfunction was successfully constructed. Two therapeutic genes, suppressor of cytokine signaling 3 (SOCS3) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were chosen and incorporated into this vector system, respectively. The combined treatment of AFP-D55-SOCS3 and AFP-D55-TRAIL (2:3 ratio) exhibited potent antitumor activity in AFP-positive HCC cell lines compared with any other treatment both in vitro and in vivo. Specific replication and low progeny yield in AFP-positive HCC cell lines rendered these double-regulated oncolytic adenoviruses remarkably safe. Our data demonstrated that restoration of SOCS3, which inhibits the JAK/STAT3 pathway, by AFP-D55-SOCS3 not only could antagonize HCC therapeutic resistance to TRAIL and adenoviruses, but could also induce cell cycle arrest in HCC cell lines. SOCS3 could down-regulate Cyclin D1 and anti-apoptotic proteins such as XIAP, Survivin, Bcl-xL, and Mcl-1, which are responsible for the synergistic inhibitory effects of AFP-D55-SOCS3 and AFP-D55-TRAIL. Dual gene and double-regulated oncolytic adenoviruses may provide safety and excellent antitumor effects for liver cancer, which is the advantage of a cancer-targeting gene virotherapy strategy.

Oncolytic adenoviruses may offer a new treatment option and improve the prognosis for patients with hepatocellular carcinoma (HCC). However, the antitumor efficacy of oncolytic adenoviruses on HCC cells is compromised due to low expression of the adenovirus serotype 5 (Ad5) receptor on the target cells. In this study we showed that all HCC cell lines and clinical samples expressed high level of CD46, the receptor for Adenovirus 35 (Ad35) and constructed new fiber chimeric oncolytic adenoviruses with or without a p53 gene expression cassette, SG635-p53 and SG635, respectively. These variants were derived from the previously described Ad5 vectors SG600-p53 and SG600 by replacing the Ad5 fiber with a chimeric Ad5/35 fiber. It was found that the 5/35 fiber chimeric adenovirus vector (Ad5/35-EGFP) demonstrated significantly improved transduction in all tested HCC cell lines compared with the Ad5 vector (Ad5-EGFP). The new fiber chimeric oncolytic adenoviruses produced more progeny viruses in HCC cells than did the Ad5-based viruses but replicated weakly in normal fibroblast BJ cells. In addition, SG635-p53 mediated a higher level of transgenic expression than SG600-p53 in Hep3B and Huh7 cells and showed a markedly enhanced antitumor effect on HCC cells in vitro compared with SG635 or SG600-p53 without causing significant cytotoxicity to normal cells. Antitumor activity of SG635-p53 was shown in Hep3B subcutaneous xenograft tumor models following intratumoral injection, resulting in significant inhibition of tumor growth and prolonged survival of animals. Our data suggest that SG635-p53, as a fiber chimeric oncolytic adenovirus in combination with p53 expression, may serve as a novel, promising and safe anticancer agent for the treatment of HCC.

Using conditionally replicating adenoviral vectors (CRAds) is a promising strategy in the treatment of solid tumors. The prospective of this study was to design a novel CRAd for the treatment of gastrointestinal cancer and show its efficacy in vitro, as well as in vivo. To determine if aberrant wnt signaling in tumor cells can be used to selectively drive viral replication, we analyzed six colorectal and hepatocellular cell lines, as well as 13 colorectal tumors and 17 gastric tumors, for β-catenin mutation status or aberrant wnt signaling, both of which were found frequently. Based on these findings, a novel CRAd (Ad5F11.wnt-E1A-hIL24) containing an E1A expression cassette driven by an artificial wnt promoter and delivering an apoptosis-inducing gene, interleukin-24 (IL24), was engineered. To enhance infection efficiency, the virus was pseudotyped by replacing adenovirus serotype 5 (Ad5) with Ad11 fiber. Ad5F11.wnt-E1A-hIL24 virus exhibited high selectivity toward cells with aberrant wnt signaling both in vitro and in mouse xenograft tumors. Transduction efficiency was significantly improved compared with that of nonpseudotyped control viruses. The proliferation of tumor cell lines, as well as tumor growth, in mouse xenografts could be profoundly inhibited by viral infection with Ad5F11.wnt-E1A-hIL24. The therapeutic effect was associated with increased apoptosis through caspase-3 activation. In addition, Ad5F11b vector exhibited a more favorable biodistribution, blood clearance, and transgene expression compared with conventional Ad5 vector after systemic or intratumoral injection in human gastrointestinal cancer xenografts. We think that our approach is a promising strategy in the treatment of gastrointestinal cancer, warranting further clinical investigation.

Conditionally replicative adenoviruses (CRAds) are widely used for cancer biotherapy and show a significant growth-suppressing effect on many types of cancer. However, it was reported that breast cancer was highly resistant to the infection of traditionally used adenovirus of serotype 5 (Ad5)-based CRAds. Although partial substitution of the fiber protein of replication-deficient Ad5 with that of adenovirus of serotype 35 (Ad35) facilitated infection of breast cancer cells by adenoviral vectors, it is still unknown whether this modification can improve CRAds in their tumor-eliminating capacity. We generated a 5/35 fiber-modified CRAd with a p53 cDNA construct and investigated whether this alteration in fiber region can make CRAds suppress the growth of breast cancer more effectively. Our data reinforced the proposal that 5/35-modified fiber conferred higher adenovirus infectivity for breast cancer cells than natural Ad5 fiber. Interestingly, 5/35 fiber-modified CRAd replicated more efficiently in breast cancer cells than Ad5-based CRAd. We also found 5/35 fiber-modified CRAd mediated higher expression of p53 in breast cancer cells. In vitro, 5/35 fiber-modified CRAd eliminated breast cancer cells more efficiently. Growth of xenograft tumors in nude mice was also significantly retarded by 5/35 fiber-modified CRAd. The 5/35 fiber-modified CRAd suppressed the growth of breast cancer cells more effectively than Ad5-based CRAd, both in vitro and in vivo. Thus CRAd with 5/35 hybrid fiber may be a promising vector for breast cancer treatment.

Background Conditionally replicative adenovirus (CRAd) provides a promising strategy for solid tumor therapy. However, relatively few studies have been addressed on hematopoietic malignancies. We previously found that ZD55, a serotype 5 (Ad5)-based, E1B 55-kDa deleted CRAd, inhibited leukemic cell growth and induced apoptosis. In the present study, we employed SG235, a new CRAd with both an E1B 55-kDa deletion and an Ad5/F35 chimeric fiber, for the treatment of B-cell tumors. Methods CRAd SG235 was engineered not to express adenovirus E1B 55-kDa gene, and the wild-type Ad5 fiber was replaced by a chimeric Ad5/35 fiber containing an Ad5 tail, an Ad35 shaft and an Ad35 knob. Using in vitro and in vivo experiments, the infectivity and selective cytotoxicity of SG235 on B-cell tumor lines were evaluated. Apoptosis-related signaling elements were investigated. Results SG235 significantly suppressed malignant B-cell growth in vitro and in vivo. In addition to selective cytolysis, SG235-induced apoptosis in the tumor cells. Upon SG235 infection, levels of cleaved forms of caspase-3 and poly(adenosine diphosphate-ribose) polymerase increased, suggesting that SG235 induces apoptosis in malignant B-cells by activating a caspase cascade. Furthermore, SG235 infection resulted in an up-regulated level of Bax, as well as down-regulated levels of xIAP, cIAP and survivin, suggesting that infection of SG235 induces apoptosis in B-cell tumor lines by affecting both apoptosis-promoting and -inhibiting intracellular signaling elements. Conclusions CRAd SG235 may serve as a potential anticancer agent, or a therapeutic vehicle for harboring anticancer genes, in B-cell tumor treatment. Copyright © 2009 John Wiley & Sons, Ltd.

Conditionally replicating adenoviruses (CRAd) have been under extensive investigations as anticancer agents. Previously, we found that ZD55, an adenovirus serotype 5-based CRAd, infected and killed the leukemia cells expressing coxsackie adenovirus receptor (CAR). However, majority of leukemic cells lack CAR expression on their cell surface, resulting in resistance to CRAd infection. In this study, we showed that SG235, a novel fiber chimeric CRAd that has Ad35 tropism, permitted CAR-independent cell entry, and this in turn produced selective cytopathic effects in a variety of human leukemic cells in vitro and in vivo. Moreover, SG235 expressing exogenous tumor necrosis factor-related apoptosis-inducing ligand (SG235-TRAIL) effectively induced apoptosis of leukemic cells via the activation of extrinsic and intrinsic apoptotic pathway and elicited a superior antileukemia activity compared with SG235. In addition, normal hematopoietic progenitors were resistant to the inhibitory activity of SG235 and SG235-TRAIL. Our data suggest that these novel oncolytic agents may serve as useful tools for the treatment of leukemia. [Mol Cancer Ther 2009;8(5):1387–97]

Targeting is critical for successful tumor gene therapy. The adeno-associated virus (AAV) has aroused wide concern due to its excellent advantages over other viral vectors in gene therapy. AAV has a broad infection spectrum, which also results in poor specificity towards tissues or cells and low transduction efficiency. Therefore, it is imperative to improve target and transduction efficiency in AAV-mediated gene therapy. Up to now, researchers have developed many strategies to modify AAV capsids for improving targeting or retargeting only desired cells. These strategies include not only traditional chemical modification, phage display technology, modification of AAV capsid genome, chimeric vectors and so on, but also many novel strategies involved in marker rescue strategy, direct evolution of capsid proteins, direct display random peptides on AAV capsid, AAVP (AAV-Phage), and etc. This review will summarize the advances of researches on the capsid modification of AAV to target malignant cells.

Cancer stem cell/tumor-initiating cell (CSC/TIC) is a subclass of cancer cells possessing parts of properties of normal stem cell. It has a high capacity of proliferation and plays a pivotal role in tumor recurrence and tumor resistance to radiotherapy and chemotherapy. At present, small molecule inhibitors and fusion proteins are widely used in the CSC-targeting strategy. Gene-virotherapy, which uses oncolytic adenovirus as a vector to mediate the expression of therapeutic gene, shows a significant superiority to other regimens of cancer treatment and has a good efficacy in the treatment of solid tumors. Thus, it is a promising choice to apply gene-virotherapy into the CSC-targeting treatment. Based on the molecular mechanism underlying CSC self-renewal, a series of effective strategies for targeting CSC have been established. This review will summarize the recent research progresses on CSC-targeting treatment.

Conditionally replicative adenovirus (CRAD) represents a promising approach for cancer therapy. Several CRADs controlled by the human telomerase reverse transcriptase promoter have been developed. However, because of their replicative capacity, the importance of cancer specificity for CRADs needs to be further emphasized. In this study, we have developed a novel dual-regulated CRAD, CNHK500-mE, which has its E1a and E1b gene controlled by the human telomerase reverse transcriptase promoter and the hypoxia response element, respectively. It also carries a mouse endostatin expression cassette controlled by the cytomegalovirus promoter. These properties allow for increased cancer cell targeting specificity and decreased adverse side effects. We showed that CNHK500-mE preferentially replicated in cancer cells. Compared with a replication-defective vector carrying the same endostatin expression cassette, CNHK500-mE–mediated transgene expression level was markedly increased via viral replication within cancer cells. In the nasopharyngeal tumor xenograft model, CNHK500-mE injection resulted in antitumor efficacy at day 7 after therapy. Three weeks later, it led to significant inhibition of xenograft tumor growth due to the combined effects of viral oncolytic therapy and antiangiogenesis gene therapy. Pathologic examination showed that most cancer cells were positive for adenoviral capsid protein and for apoptotic terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling in the CNHK500-mE–treated tumor tissues, and the microvessels in these tumor tissues were diminished in quantity and abnormal in morphology. These results suggest that, as a potential cancer therapeutic agent, the CNHK500-mE is endowed with higher specificity to cancer cells and low cytotoxicity to normal cells. (Mol Cancer Res 2008;6(4):568–75)

Since the first parvovirus serotype AAV2 was isolated from human and used as a vector for gene therapy application, there have been significant progresses in AAV vector development. AAV vectors have been extensively investigated in gene therapy for a broad application. AAV vectors have been considered as the first choice of vector due to efficient infectivity, stable expression and nonpathogenicity. However, the untoward events in AAV mediated in vivo gene therapy studies proposed the new challenges for their further applications. Deep understanding of the viral life cycle, viral structure and replication, infection mechanism and efficiency of AAV DNA integration, in terms of contributing viral, host-cell factors and circumstances would promote to evaluate the advantages and disadvantages and provide more insightful information for the possible clinical applications. In this review, main effort will be focused on the recent progresses in gene delivery to the target cells via receptor-ligand interaction and DNA specific integration regulation. Furthermore AAV receptor and virus particle intracellular trafficking are also discussed.