B7-H3 targeted CAR-T cells show highly efficient anti-tumor function against osteosarcoma both in vitro and in vivo.

BACKGROUND: Osteosarcoma (OS) mainly happens in children and youths. Surgery, radiotherapy and chemotherapy are the common therapies for osteosarcoma treatment but all their anti-tumor effects are limited. In recent years, a new cellular therapy, CAR-T, a cellular immunotherapy with genetically engineered T cells bearing chimeric antigen receptor targeting specific tumor-associated antigen, has been proved to be an effective therapy against acute lymphoblastic leukemia. Thus, CAR-T is a potentially effective therapy for osteosarcoma treatment. METHODS: A CAR gene targeting B7-H3 antigen was constructed into lentiviral vector through molecular biology techniques. Then, the CAR gene was transferred to T cells through lentiviral delivery system, and the CAR-T cells were largely expanded using in vitro culture technology. The in vitro anti-tumor effect of CAR-T cells was evaluated through Real Time Cell Analysis system (RTCA) and ELISA assay. The in vivo anti-tumor capabilities of CAR-T cells were evaluated using the patient-derived xenografts (PDX) model of osteosarcoma. RESULTS: The third-generation CAR-T cells we constructed could target the B7-H3 antigen, and the phenotype of CAR-T cells was consistent with normal T cells; The CAR-T cells showed superior antitumor effects both in vitro and in vivo. CONCLUSION: Our study showed that B7-H3 targeted CAR-T cells had high anti-tumor efficacy against osteosarcoma both in vitro and in vivo, which proved that B7-H3 targeted CAR-T therapy is potentially effective for osteosarcoma treatment.

Detection of residual E. coli host cell DNA by 23S ribosomal RNA gene-targeted quantitative polymerase chain reactions.

Among the many systems available for heterologous protein production gram-negative bacterium Escherichia coli (E. coli) has long been widely used because of its ability to grow rapidly with a high density on inexpensive substrates. The use of E. coli as the host system has many regulatory issues, one of which is the residual host cell DNA. Residual DNA carried by biological products may lead to carcinogenicity and immunomodulation risks. The World Health Organization (WHO) for the acceptable amounts of residual host cell DNA is less than 10 ng per dose. Therefore, it is important to keep an extremely low level of residual host DNA in the biological products derived from E. coli. In this study, we designed primer/probe sets targeting E. coli 23S ribosomal RNA gene to quantify the residual DNA of E. coli by quantitative polymerase chain reactions (qPCR). Result showed that this primer/probe has high species specificity. The limit of detection (LOD) in this method is 0.01 pg/µl and this allowed for detection of residual host DNA of much lower concentrations. We assessed accuracy by calculating the recovery (92.1∼140.1 %) of the spiked DNA in plasmids which were produced from E. coli. We also checked intra-assay precision (9.8∼15.1 %) and inter-assay precision (10.9∼18.3 %) by repeatedly measuring the four different concentration standards. In addition, the robustness assay was performed by generating standard curve using short length E. coli DNA. The result showed that appropriate degree of DNA fragmentation will not affect tests. These validation studies demonstrated that our method has excellent specificity, linearity, accuracy, precision and robustness.

The antitumor capacity of mesothelin-CAR-T cells in targeting solid tumors in mice.

Since the approval of chimeric antigen receptor (CAR) T cell therapy targeting CD19 by the FDA, CAR-T cell therapy has received increasing attention as a new method for targeting tumors. Although CAR-T cell therapy has a good effect against hematological malignancies, it has been less effective against solid tumors. In the present study, we selected mesothelin (MSLN/MESO) as a target for CAR-T cells because it is highly expressed by solid tumors but only expressed at low levels by normal tissues. We engineered a third generation MSLN-CAR comprising a single-chain variable fragment (scFv) targeting MSLN (MSLN-scFv), a CD8 transmembrane domain, the costimulatory domains from CD28 and 4-1BB, and the activating domain CD3ζ. In vitro, MSLN-CAR-T cells killed various solid tumor cell lines, demonstrating that it could specifically kill MSLN-positive cells and release cytokines. In vivo, we investigated the effects of MSLN-CAR-T cell therapy against ovarian, breast, and colorectal cancer cell-line-derived xenografts (CDX) and MSLN-positive colorectal and gastric cancer patient-derived xenografts (PDX). MSLN-CAR decreased the growth of MSLN-positive tumors concomitant with significantly increased T cells and cytokine levels compared to the control group. These results indicated that modified MSLN-CAR-T cells could be a promising therapeutic approach for solid tumors.

PD-1 silencing improves anti-tumor activities of human mesothelin-targeted CAR T cells.

Chimeric antigen receptor T (CAR T) cell therapy is a new pillar in cancer therapeutics, and has been successfully used for the treatment of cancers, including acute lymphoblastic leukemia and solid cancers. Following immune attack, many tumors upregulate inhibitory ligands which bind to inhibitory receptors on T cells. For example, the interaction between programmed cell death protein 1 (PD-1) on activated T cells and its ligands (widely known as PD-L1) on a target tumor limits the efficacy of CAR T cells therapy against poorly responding tumors. Here, we use mesothelin (MSLN)-expressing human ovarian cancer cells (SKOV3) and human colon cancer cells (HCT116) to investigate whether PD-1-mediated T cell exhaustion affects the anti-tumor activity of MSLN-targeted CAR T cells. We utilized cell-intrinsic PD-1-targeting shRNA overexpression strategy, resulting in a significant PD-1 silencing in CAR T cells. The reduction of PD-1 expression on T cell surface strongly augmented CAR T cell cytokine production and cytotoxicity towards PD-L1-expressing cancer cells in vitro. This study indicates the enhanced anti-tumor efficacy of PD-1-silencing MSLN-targeted CAR T cells against several cancers and suggests the potential of other specific gene silencing on the immune checkpoints to enhance the CAR T cell therapies against human tumors.