The effects of aging, sex, and tumor burden on the peripheral blood immune cell profile and absolute counts.

Background: To better assess the peripheral immune status and aid in the early diagnosis and prognosis of tumors, we compared the proportion and absolute counting of peripheral immune cell subsets in healthy individuals and tumor patients of varying ages, taking into account the impact of sex and tumor metastasis. Methods: We used peripheral blood mononuclear cell (PBMC) samples from 520 patients with various tumor types and 109 healthy volunteers. The absolute numbers of lymphocytes and monocytes were identified by an automated blood analyzer, and multi-parameter flow cytometry was used to examine the subsets of natural killer (NK) cells (CD3-CD16+CD56+), T cells (CD3+CD4+/CD8+), and mononuclear cells (CD14+) in PBMC. Results: The percentage of T cells (CD3+) in peripheral blood mononuclear cells (PBMC) was 55.83% VS 45.54% (P<0.0001) between healthy volunteers and tumor patients, showing a significant downward trend. Meanwhile, the percentages of monocytes (CD14+) and NK cells (CD3-CD16+CD56+) showed a significant upward trend. Single factor or multifactor analysis yielded identical findings on the proportion of PBMC between healthy individuals and patients with different malignancies, considering the three confounding variables of age, sex, and tumor metastasis. Conclusion: The proportion and absolute counting of acquired immune T cells, innate immune NK cells, and monocytes in PBMCs all exhibit substantial changes between cancer patients and healthy individuals, and the differences are influenced by age, sex, and tumor progression.

Antitumor activity of Z15-0-2, a bispecific nanobody targeting PD-1 and CTLA-4.

The combination of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) antibodies has potential for enhancing clinical efficacy. We described the development and antitumor activity of Z15-0, a bispecific nanobody targeting both the PD-1 and CTLA-4 pathways simultaneously. We designed and optimized the mRNA sequence encoding Z15-0, referred to as Z15-0-2 and through a series of in vitro and in vivo experiments, we established that the optimized Z15-0-2 mRNA sequence significantly increased the expression of the bispecific nanobody. Administration of Z15-0-2 mRNA to tumor-bearing mice led to greater inhibition of tumor growth compared to controls. In aggregate, we introduced a novel bispecific nanobody and have re-engineered it to boost expression of mRNA, representing a new drug development paradigm.

Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system.

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs_0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO-K1 cells. The results showed that Rs_0636/Rs_0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs_0636/Rs_0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines.

Rapid response in relapsed follicular lymphoma with massive chylous ascites to anti-CD19 CAR T therapy using Piggy Bac: A case report.

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 Registration: https://ClinicalTrials.gov, identifier NCT05472610.

Horizontal Transfer and Evolutionary Profiles of Two Tc1/DD34E Transposons (ZB and SB) in Vertebrates.

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.

Single-cell RNA-Seq reveals the potential risk of anti-mesothelin CAR T Cell therapy toxicity to different organs in humans.

"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 (<1%). Therefore, these organs could be regarded as at low risk. Thus, the risk of toxicity to different organs and tissues in anti-mesothelin CAR T cell therapy may be predicted by these scRNA-seq data.

The role of mesenchymal stem cells in liver injury.

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.

PiggyBac-Generated CAR19-T Cells Plus Lenalidomide Cause Durable Complete Remission of Triple-Hit Refractory/Relapsed DLBCL: A Case Report.

MYC/BCL2/BCL6 triple-hit lymphoma (THL) is an uncommon subset of high-grade B-cell lymphoma with aggressive clinical behavior and poor prognosis. TP53 mutation is an independently poor progonistic indicator in patients with THL, hence novel therapeutic strategies are needed for these patients. CD19-directed chimeric antigen receptor(CAR19)-T cell therapy has shown promising efficacy for relapsed/refractory diffuse large B cell lymphoma (RR DLBCL), but the majority of CAR19-T cell products to date have been manufactured using viral vectors. PiggyBac transposon system, with an inclination to memory T cells, offers a more convenient and economical alternative for transgene delivery. We herein report the first case of triple-hit RR DLBCL with TP53 mutation who was treated with piggyBac-generated CAR19-T cells and accompanied by grade 2 cytokine release syndrome. The patient obtained a complete remission (CR) in the 2nd month post-infusion and demanded maintenance therapy. Whether maintenance therapy is favorable and how to administrate it after CAR-T cell infusion remain controversial. Preclinical studies demonstrated that lenalidomide could enhance antitumor activity of CAR19-T cells. Therefore, we pioneered oral lenalidomide after CAR19-T therapy in the patient from the 4th month, and he discontinued after one cycle due to side effects. The patient has still kept sustained CR for over 24 months. Our case have firstly demonstrated the feasibility, preliminary safety and efficacy of piggyBac-produced CAR19-T cell therapy in triple-hit lymphoma. The innovative combination with lenalidomide warrants further investigation. Our findings shed new light on the possible solutions to improve short-term relapse after CAR19-T cell therapy in RR DLBCL. ChiCTR, number ChiCTR1800018111.

Phase I clinical trial of EGFR-specific CAR-T cells generated by the piggyBac transposon system in advanced relapsed/refractory non-small cell lung cancer patients.

PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Chimeric antigen receptor T cells engineered to secrete CD40 agonist antibodies enhance antitumor efficacy.

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.

αPD-1-mesoCAR-T cells partially inhibit the growth of advanced/refractory ovarian cancer in a patient along with daily apatinib.

Epithelial ovarian cancer (EOC) is the leading cause of death among gynecological malignancies in China. In particular, advanced/refractory ovarian cancer lacks effective targeted therapies due to the immunosuppressive and proangiogenic tumor microenvironment. Mesothelin (MSLN) has been found to be highly expressive in most EOC. Targeting MSLN by antibodies or chimeric antigen receptor-modified T (CAR-T) cells and immune checkpoint blockades as well as apatinib, an anti-angiogenic drug, have been used in patients with refractory ovarian cancer. Apatinib was reported to promote the infiltration of CD8+ T cells in lung cancer. However, the combination therapy of CAR-T secreting anti-PD-1 antibody with apatinib in EOC has not been reported. CASE PRESENTATION: Here we report a case of refractory EOC in a patient who had relapsed after multiline chemotherapy. The patient received autologous T cells that contained sequences encoding single-chain variable fragments specific for MSLN and full-length antibody for PD-1 (αPD-1). The modified T cells were called αPD-1-mesoCAR-T cells. After infusion, the copy number and PD-1 antibody secretion of the CAR-T cells were increased in the blood. By application of multimodality tumor tracking, MRI of the liver showed shrinkage of metastatic nodules from average diameter of 71.3-39.1 mm at month 2. The patient achieved partial response and survived more than 17 months. IL-6 levels in the patient fluctuated from the baseline to 2-4-folds after treatment, but side effects were mild with only grade 1 hypertension and fatigue. CONCLUSION: αPD-1-mesoCAR-T cell therapy combined with apatinib demonstrates a potential therapeutic effect on advanced refractory ovarian cancer. TRIAL REGISTRATION NUMBER: NCT03615313.

Current Progress in CAR-T Cell Therapy for Hematological Malignancies.

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.

Evaluation of Nonviral piggyBac and lentiviral Vector in Functions of CD19chimeric Antigen Receptor T Cells and Their Antitumor Activity for CD19+ Tumor Cells.

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.

Safety and Efficacy of an Immune Cell-Specific Chimeric Promoter in Regulating Anti-PD-1 Antibody Expression in CAR T Cells.

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.

An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications.

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.

Triggered Immune Response Induced by Antigenic Epitopes Covalently Linked with Immunoadjuvant-Pulsed Dendritic Cells as a Promising Cancer Vaccine.

The success of peptide-based dendritic cell (DC) cancer vaccines mainly depends on the utilized peptides and selection of an appropriate adjuvant. Herein, we aimed to evoke a broad immune response against multiple epitopes concurrently in the presence of immunoadjuvant. Three synthetic HLA-A∗0201-restricted peptides were separately linked with HMGB1-derived peptide (SAFFLFCSE, denoted as HB100-108) as immunoadjuvant via double arginine (RR) linker and loaded onto human monocyte-derived DCs. Peptide uptake was detected by immunofluorescence microscopy and flow cytometry. The maturation and activation status of pulsed DCs were monitored by detection of the expression of specific markers and released cytokines. The ability of peptide-pulsed DCs to activate allogeneic T cells has been assessed by a degranulation assay and detection of secreted cytokines. The lytic activity of effector T cells against cancer cells in vitro was analyzed by a lactate dehydrogenase (LDH) assay. Results revealed that DCs efficiently take up peptides+HB100-108 and expressed higher levels of surface markers (HLA-ABC, HLA-DR, CD80, CD86, CD83, CD40, and CCR7) and proinflammatory cytokines (IL-6, IFN-γ, TNF-α, and IL-12) than control DCs, free peptide-pulsed DCs, and free HB100-108-pulsed DC groups. Moreover, peptides+HB100-108/pulsed DCs were capable of activating allogeneic T cells and enhance their lytic activity against a pancreatic cancer cell line (PANC-1) in vitro. These findings suggest that antigenic peptides covalently linked with HB100-108/pulsed DCs could be a promising strategy to improve the current DC-based cancer vaccines.

Correction: Modified CAR T cells targeting membrane-proximal epitope of mesothelin enhances the antitumor function against large solid tumor.

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

Changes and Clinical Significance of Detailed Peripheral Lymphocyte Subsets in Evaluating the Immunity for Cancer Patients.

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<0.01), Naïve Tc cells (p<0.01), CM (central memory) Tc cells (p<0.01), Naïve T cells/Memory T cells (p<0.001), Naïve T cells/EM (effector memory) T cells (p<0.001) and Naive Th cells/Memory Th cells (p< 0.001), and higher proportion and ratio of EM Th cells (p<0.001), EM Tc cells (p<0.01), effector Tc cells (p<0.05), EM Th cells/CM Th cells (p<0.01) and EM Tc cells/CM Tc cells (p<0.01), as well as Breg (p<0.001), B cells (p<0.05) and CD16-NK cells (p<0.001) were found in cancer cohorts. CONCLUSION: This study suggests that the changes in certain lymphocyte subsets might be helpful to evaluate the immunity of cancer patients, and holds great potential for clinical application.

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.

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.