Rapid generation of CD19 CAR-T cells by minicircle DNA enables anti-tumor activity and prevents fatal CAR-B leukemia.

Manufacturing chimeric antigen receptor (CAR)-T cells using viral vectors is expensive and time-consuming. In addition, during viral transduction, genes encoding CARs are randomly integrated into the genome, which can cause oncogenesis or produce devastating CAR-tumor cells. Here, using a virus-free and non-transgenic minicircle DNA (mcDNA) vector, we enabled the rapid generation of CD19 CAR-T cells within two days. Furthermore, we demonstrated in vitro and in xenograft models that the antitumor effects of CD19 CAR-T cells produced by mcDNA are as effective as those produced by viral vectors. Finally, we showed that our manufacturing process avoids the production of fatal CAR-tumor cells. Taken together, we have provided a fast, effective, and therapeutically safe method for generating CD19 CAR-T cells for the treatment of leukemia.

Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) has been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like "off-target" toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment. METHODS: The peripheral blood was acquired from healthy donors and T cells were separated by density-gradient centrifugation. We used an electroporation system to deliver anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures as target genes into the T cells. The cell membrane was opened by the momentary electric current effect, and the target gene was delivered into the cell by non-viral minicircle DNA (mcDNA) vector. The flow cytometry and western blot assays were used to detect whether the two scFv were simultaneously transfected and the transfection efficacy of this bispecific CAR T cell generation method. We respectively detected the in vitro and in vivo tumor-suppression efficacy of CAR T cells through the CCK-8 assays and the HCC xenograft mice models. The CoG133-CAR T cells containing both CD133 and GPC3 antigen recognition sites were the effector cells. CD133-CAR T cells and GPC3-CAR T cells were defined as single-targeted control groups, normal T and mock T cells were defined as blank control groups. RESULTS: The mcDNA vector accommodated two target gene structures successfully transfected to generate bispecific CAR T cells. The detection methods on gene level and protein level confirmed that CoG133-CAR T cells had considerable transfection efficiency and exhibited both antigen-binding capacity of CD133 and GPC3. Compared to single-targeted CAR T cells or control T cells, CoG133-CAR T cells performed enhanced eliminated efficacy against CD133 and GPC3 double-positive HCC cell line in vitro and HCC xenograft mice in vivo. Hematoxylin and eosin (H&E) staining indicated no fatal "off-target" combination existed on CoG133-CAR T cells and major organs. CONCLUSION: Our study suggests that it is with higher efficiency and more safety to prepare bispecific CAR T cells through non-viral mcDNA vectors. CoG133-CAR T cells have enhanced tumor-suppression capacity through dual antigen recognition and internal activation. It provides an innovative strategy for CAR T therapy of HCC, even solid tumors.

Anti-HER2 scFv-CCL19-IL7 recombinant protein inhibited gastric tumor growth in vivo.

HER-2 targeted therapies, such as monoclonal antibodies (mAbs) and CAR-T cell therapy have been applied in the treatment of various of cancers. However, the anti-HER2 CAR-T cell therapy are limited by its expensive production procedure and fatal side effects such as cytokine storm or "On target, off tumor". The application of anti-HER2 mAbs to the soild tumor are also plagued by the patients resistant with different mechanisms. Thus, the recombinant protein technology can be presented as an attractive methods in advantage its less toxic and lower cost. In this study, we produced a HER-2-targeting recombinant protein, which is the fusion of the anti-HER-2 single chain fragment variable domain, CCL19 and IL7 (HCI fusion protein). Our results showed that the recombinant protein can induce the specific lysis effects of immune cells on HER-2-positive gastric tumor cells and can suppress gastric tumor growth in a xenograft model by chemotactic autoimmune cell infiltration into tumor tissues and activated T cells. Taken together, our results revealed that the HCI fusion protein can be applied as a subsequent clinical drug in treating HER-2 positive gastric tumors.

Folate Receptor-Alpha Targeted 7x19 CAR-γδT Suppressed Triple-Negative Breast Cancer Xenograft Model in Mice.

Background: Triple-negative breast cancer (TNBC) is the worst prognosis subtype of breast cancer due to lack of specific targets. Recent studies have shown that immunotherapy may solve that problem by targeting folate receptor-alpha (FRα). Methods: Gene modified γδ T cells were manufactured to express FRa specific chimeric antigen receptor (FRa CAR) and secrete interleukin-7 (IL-7) and chemokine C-C motif ligand 19 (CCL19). CAR-γδT cells that secrete IL-7 and CCL19 (7 × 19 CAR-γδT) were evaluated for their antitumor activity both in vitro and in vivo. Results: 7 × 19 CAR-γδT showed remarkable antitumor activity in vitro. Combined with PBMC, 7 × 19 CAR-γδT inhibited TNBC xenograft model growth superiorly compared with single-application or conventional CAR-γδT cells. Histopathological analyses showed increased DC or T cells infiltration to tumor tissues. Conclusion: Taken together, our results showed that 7 × 19 CAR-γδT have remarkable anti-TNBC tumor activity and showed a broad application prospect in the treatment of incurable TNBC patients.

Therapeutic effects of CXCL9-overexpressing human umbilical cord mesenchymal stem cells on liver fibrosis in rats.

Umbilical cord mesenchymal stem cells (UC-MSCs) transplantation has become a promising treatment for liver fibrosis. However, UC-MSCs have limited anti-fibrosis ability, and their homing ability of UC-MSCs to the injured liver seems to be poor. In our study, we aimed to determine if the CXCL9-overexpressing UC-MSCs could have synergistic anti-fibrosis effects and whether it can promote the homing ability of UC-MSCs. Overexpression of CXCL9 in UC-MSCs (CXCL9-UC-MSCs) was attained by transfecting the lenti-CXCL9-mCherry to naive UC-MSCs. The therapeutic effect of transducted CXCL9-UC-MSCs on both repairing of hepatic fibrosis and target homing were evaluated by comparing with the control of UC-MSCs transfected with empty lenti-mCherry vector. The results revealed that the liver function of CXCL9-UC-MSCs treated group was significantly improved when compared with that of control UC-MSCs (P < 0.05), and the histopathology indicated an obvious decrease of the collagen fiber content and significant disappearing of pseudo-lobules with basically normal morphology of hepatic lobules. Furthermore, liver frozen sections confirmed that CXCL9-UC-MSCs have significantly stronger chemotaxis and stable persistence in the injured liver tissues. In summary, overexpression of CXCL9 could improve the efficacy of UC-MSCs therapy for liver fibrosis repairing on account of an enhanced ability of UC-MSCs in homing to and staying in the injured sites of liver fibrosis in rat models.

Minicircle DNA-Mediated CAR T Cells Targeting CD44 Suppressed Hepatocellular Carcinoma Both in vitro and in vivo.

PURPOSE: Based on the continuous exploration of solid tumor immunotherapy, we focused on hepatocellular carcinoma with a high level of morbidity and mortality. We confirm the stability of mcDNA-based CAR T cell generating platform, and investigate the antitumor activity of CD44-CAR T cells against hepatocellular carcinoma both in vitro and in vivo. MATERIALS AND METHODS: We fused anti-CD44 scFv structure with transmembrane domain and intracellular domain. Using a non-viral mcDNA vector to load CD44-CAR gene, then transfected the mcDNA-CD44-CAR into human T cells by electroporation. We exhibited the transfection efficacy of CAR T cells and the CD44 expression of tumor cell lines by flow cytometry. The antitumor efficacy of CD44-CAR T cells in vitro and in vivo was detected through CCK-8 and ELISA assays, and xenograft mouse models, respectively. RESULTS: We obtained mcDNA-CD44-CAR with a high level of density after repeated extraction and purification. The expression efficacy of CD44-CAR in T cells was more than 50% after seven days electroporation and the phenotype of CD44-CAR T cells was no difference compared with normal T cells. For CD44-positive hepatocellular carcinoma xenograft mice, CD44-CAR T cells had stronger tumor growth suppression compared to normal T and mock T cells. The same results occurred on the in vitro experiments including cytokine secretion and cytotoxicity assays. H&E staining graphs revealed that CD44-CAR T cells did not induce side effects in xenograft mice. CONCLUSION: The strategy for generating CAR T cells targeting cancer stem cell antigens was efficient and concise. The mcDNA had superior transgene ability without virus-related adverse effects. CD44-CAR T cells had strong suppression capacity against hepatocellular carcinoma.

Minicircle DNA-Engineered CAR T Cells Suppressed Tumor Growth in Mice.

Viral-based chimeric antigen receptor-engineered T (CAR T)-cell manufacturing has potential safety risks and relatively high costs. The nonviral minicircle DNA (mcDNA) is safer for patients, cheaper to produce, and may be a more suitable technique to generate CAR T cells. In this study, we produced mcDNA-based CAR T cells specifically targeting prostate stem cell antigen (PSCA; mcDNA-PSCA-CAR T cells). Our results showed that mcDNA-PSCA-CAR T cells persisted in mouse peripheral blood as long as 28 days and demonstrated more CAR T-cell infiltration, higher cytokine secretion levels, and better antitumor effects. Together, our results suggest that mcDNA-CAR can be a safe and cost-effective platform to produce CAR T cells.

Disruption of CTLA-4 expression on peripheral blood CD8 + T cell enhances anti-tumor efficacy in bladder cancer.

Activation of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) on T cells leads to T cell exhaustion and ultimately facilitates tumor progression. Recent success of using immune cell checkpoint inhibitors offers a great promise to treat various cancers, including bladder cancer. However, the expression pattern and therapeutic value of PD-1 and CTLA-4 in peripheral blood T cells remain largely unexplored. In this study, we presume that disruption of the potential dysregulated checkpoint molecules in peripheral blood T cells may improve the anti-tumor efficacy of cytotoxic T cells in bladder cancer. We showed that both PD-1 and CTLA-4 expression were specifically elevated on CD8 + T cells but not CD4 + T cells in peripheral blood of patients with bladder cancer compared with that in healthy donors. Notably, CTLA-4 expression was significantly higher in muscle-invasive bladder cancer (MIBC) and correlated with tumor size. By blocking CTLA-4 with anti-CTLA-4 antibody and CRISPR-Cas9-mediated CTLA-4 disruption, we revealed that CTLA-4-disrupted CTLs had enhanced cellular immune response and superior cytotoxicity to the CD80/CD86-positive bladder cancer cells in vitro. Moreover, the CTLA-4-disrupted CTLs exhibited a pronounced anti-tumor effect in vivo as demonstrated by prophylactic assay and therapeutic assay in the subcutaneous xenograft model. Collectively, our findings confirm improved therapeutic efficacy of CTLA-4-disrupted CTLs and provides the potential strategy for targeting immune checkpoints to enhance the promising immunotherapy.