NK-92MI Cells Engineered with Anti-claudin-6 Chimeric Antigen Receptors in Immunotherapy for Ovarian Cancer.

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR-NK cell efficacy. Claudin-6 (CLDN6) has been reported to be overexpressed in ovarian cancer and may be an attractive target for CAR-NK cells immunotherapy. However, the feasibility of using anti-CLDN6 CAR-NK cells to treat ovarian cancer remains to be explored. Methods: CLDN6 expression in primary human ovarian cancer, normal tissues and cell lines were detected by immunohistochemistry and western blot. Two types of third-generation CAR NK-92MI cells targeting CLDN6, CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) and CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB) were constructed by lentivirus transfection, sorted by flow cytometry and verified by western blot and qPCR. OVCAR-3, SK-OV-3, A2780, Hey and PC-3 cells expressing the GFP and luciferase genes were transduced. Subcutaneous and intraperitoneal tumor models were established via NSG mice. The ability of CLDN6-CAR NK cells to kill CLDN6-positive ovarian cancer cells were evaluated in vitro and in vivo by live cell imaging and bioluminescence imaging. Results: Both CLDN6-CAR1 and CLDN6-CAR2 NK-92MI cells could specifically killed CLDN6-positive ovarian cancer cells (OVCAR-3, SK-OV-3, A2780 and Hey), rather than CLDN6 negative cell (PC-3), in vitro. CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) exhibited stronger cytotoxicity than CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB). Furthermore, CLDN6-CAR1 NK cells could effectively eliminate ovarian cancer cells in subcutaneous and intraperitoneal tumor models. More importantly, CAR-NK cells combined with immune checkpoint inhibitors, anti-PD-L1, could synergistically enhance the antitumor efficacy of CLDN6-targeted CAR-NK cells. Conclusions: These results indicate that CLDN6-CAR NK cells possess strong antitumor activity and represent a promising immunotherapeutic modality for ovarian cancer.

Remodelling of tumour microenvironment by microwave ablation potentiates immunotherapy of AXL-specific CAR T cells against non-small cell lung cancer.

The complex immunosuppressive tumour microenvironment (TME) and lack of tumour-specific targets hinder the application of chimeric antigen receptor (CAR) T cells in the treatment of solid tumours. Combining local treatment with CAR T cell immunotherapy may regulate the TME and enhance the killing potency of CAR T cells in solid tumours. Here, we show that AXL, which is highly expressed in non-small cell lung cancer (NSCLC) but not in normal tissues, might be a target for CAR T cell therapy. AXL-CAR T cells alone cause moderate tumour regression in subcutaneous and pulmonary metastatic lung cancer cell-derived xenograft models. Combination of microwave ablation (MWA) and AXL-CAR T cells have superior antitumour efficacy. MWA enhances the activation, infiltration, persistence and tumour suppressive properties of AXL-CAR T cells in AXL-positive NSCLC patient-derived xenograft tumours via TME remodelling. The combination therapy increases the mitochondrial oxidative metabolism of tumour-infiltrating CAR T cells. Combination treatment induces significant tumour suppression without observed toxicities in humanized immunocompetent mice. The synergistic therapeutic effect of MWA and AXL-CAR T cells may be valuable for NSCLC treatment.

CAR NK-92 cells targeting DLL3 kill effectively small cell lung cancer cells in vitro and in vivo.

Small cell lung cancer (SCLC) is characterized by a high relapse rate, drug tolerance, and limited treatment choices. Chimeric antigen receptor (CAR)-modified NK cells represent a promising immunotherapeutic modality for cancer treatment. However, their potential applications have not been explored in SCLC. Delta-like ligand 3 (DLL3) has been reported to be overexpressed in SCLC and may be a rational target for CAR NK immunotherapy. In this study, we developed DLL3-specific NK-92 cells and explored their potential in the treatment of SCLC. A coculture of DLL3+ SCLC cell lines with DLL3-CAR NK-92 cells exhibited significant in vitro cytotoxicity and cytokine production. DLL3-CAR NK-92 cells induced tumor regression in an H446-derived pulmonary metastasis tumor model under a good safety threshold. The potent antitumor activities of DLL3-CAR NK-92 cells were observed in subcutaneous tumor models of SCLC. Moreover, obvious tumor-infiltrated DLL3-CAR NK-92 cells were detected in DLL3+ SCLC xenografts. These findings indicate that DLL3-CAR NK-92 cells might be a potential strategy for the treatment of SCLC.

IL-7 and CCL19-secreting CAR-T cell therapy for tumors with positive glypican-3 or mesothelin.

Although chimeric antigen receptor (CAR)-engineered T cells have shown great success in the treatment of B cell malignancies, this strategy has limited efficacy in patients with solid tumors. In mouse CAR-T cells, IL-7 and CCL19 expression have been demonstrated to improve T cell infiltration and CAR-T cell survival in mouse tumors. Therefore, in the current study, we engineered human CAR-T cells to secrete human IL-7 and CCL19 (7 × 19) and found that these 7 × 19 CAR-T cells showed enhanced capacities of expansion and migration in vitro. Furthermore, 7 × 19 CAR-T cells showed superior tumor suppression ability compared to conventional CAR-T cells in xenografts of hepatocellular carcinoma (HCC) cell lines, primary HCC tissue samples and pancreatic carcinoma (PC) cell lines. We then initiated a phase 1 clinical trial in advanced HCC/PC/ovarian carcinoma (OC) patients with glypican-3 (GPC3) or mesothelin (MSLN) expression. In a patient with advanced HCC, anti-GPC3-7 × 19 CAR-T treatment resulted in complete tumor disappearance 30 days post intratumor injection. In a patient with advanced PC, anti-MSLN-7 × 19 CAR-T treatment resulted in almost complete tumor disappearance 240 days post-intravenous infusion. Our results demonstrated that the incorporation of 7 × 19 into CAR-T cells significantly enhanced the antitumor activity against human solid tumor. Trial registration: NCT03198546. Registered 26 June 2017, https://clinicaltrials.gov/ct2/show/NCT03198546?term=NCT03198546&draw=2&rank=1.

Use of chimeric antigen receptor NK-92 cells to target mesothelin in ovarian cancer.

Mesothelin (MSLN) has been reported to be overexpressed in ovarian cancer and may be an ideal target for immunotherapy. Recent studies have suggested that natural killer (NK) cells may be better chimeric antigen receptor (CAR) drivers because of their favorable innate characteristics, such as directly recognizing and killing tumor cells, resulting in a graft-versus-tumor effect but irresponsible for graft-versus-host disease (GVHD). The therapeutic effects of CAR-engineered NK cells targeting MSLN in ovarian cancer have not been evaluated. In this study, MSLN- and CD19-targeted CAR NK-92 (MSLN- and CD19-CAR NK) cells were constructed. Both MSLN- and CD19-CAR molecules were highly expressed on the surface of NK-92 cells following lentiviral gene transduction. MSLN-CAR NK cells specifically killed MSLN-positive ovarian cancer cells (OVCAR-3 and SK-OV-3), rather than MSLN-negative cells (SK-HEP-1), in vitro. Moreover, compared with parental NK-92 cells and CD19-CAR NK cells, stronger cytokine secretion was detected in MSLN-CAR NK cells cocultured with OVCAR-3 and SK-OV-3. Furthermore, MSLN-CAR NK cells effectively eliminated ovarian cancer cells in both subcutaneous and intraperitoneal tumor models; these cells also significantly prolonged the survival of intraperitoneally tumor-bearing mice. These results demonstrate that MSLN-CAR NK cells have robust specific antitumor activity, both in vitro and in vivo, suggesting that mesothelin could be a potential target for CAR NK cells and could be applied in the treatment of ovarian cancer.

Cannabidiol protects livers against nonalcoholic steatohepatitis induced by high-fat high cholesterol diet via regulating NF-κB and NLRP3 inflammasome pathway.

Cannabidiol (CBD), an abundant nonpsychoactive constituent of marijuana, has been reported previously to protect against hepatic steatosis. In this study, we studied further the functions and mechanisms of CBD on liver inflammation induced by HFC diet. Mice feeding an HFC diet for 8 weeks were applied to test the protective effect of CBD on livers. RAW264.7 cells were incubated with LPS + ATP ± CBD to study the mechanisms of the effect of CBD against inflammasome activation. We found that CBD alleviated liver inflammation induced by HFC diet. CBD significantly inhibited the nuclear factor-κappa B (NF-κB) p65 nuclear translocation and the activation of nucleotide-binding domain like receptor protein 3 (NLRP3) inflammasome both in vivo and in vitro studies, which lead to the reduction of the expression of inflammation-related factors in our studies. In addition, Inhibitor of activation of NF-κB partly suppressed the NLRP3 inflammasome activation, while adding CBD further inhibited NF-κB activation and correspondingly suppressed the NLRP3 inflammasome activation in macrophages. In conclusion, the suppression of the activation of NLRP3 inflammasome through deactivation of NF-κB in macrophages by CBD might be one mechanism of its anti-inflammatory function in the liver.