CRISPR/Cas9-mediated TGFßRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro.

BACKGROUND: CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFß are of great importance: TGFß is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. METHODS: In this study, we hypothesized that knocking out the TGFß receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFßRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFßRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells. RESULTS: Our experiments demonstrated that TGFßRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFß in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFßRII KO CAR T-cells has been recorded. TGFßRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFßRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. CONCLUSION: The TGFßRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

Phase 2 study of a high dose of 186Re-HEDP for bone pain palliation in patients with widespread skeletal metastases.

UNLABELLED: (186)Re-1-hydroxyethylidene-1,1-diphosphonate (HEDP) is an attractive radiopharmaceutical for the treatment of bone pain arising from skeletal metastatic lesions. Currently, (186)Re-HEDP is most commonly used in European countries. The aim of this study was to investigate the palliative efficacy and adverse effects of (186)Re-HEDP in patients with different types of cancers and skeletal bone pain. METHODS: Nineteen (8 male, 11 female) patients with various cancers (breast, prostate, renal cell carcinoma, colon, and neuroendocrine tumors) and painful bone metastases were included in the study. A dose of 1,480-3,330 MBq (40-90 mCi) of (186)Re-HEDP was administered intravenously. The patients' level of pain relief was assessed by the Visual Analog Scale for 8 wk after treatment and by a weekly blood cell count to evaluate for hematologic toxicity. RESULTS: The overall response rate was 89.5%, and the mean pain score assessed by the Visual Analog Scale was reduced from 9.1 to 5.3 after 1 wk (P = 0.003). No adverse effects were reported by patients during intravenous administration or for up to 24 h after administration. A flare reaction was seen in 63.2% of patients, mainly during days 1-3, and lasted for 2-4 d. There was no significant correlation between the response to therapy and the flare reactions (P > 0.05). The nadir of platelet reduction occurred at the fourth or fifth week and led to platelet infusion in only 4 patients with a low baseline platelet count and diffuse skeletal metastases. Bone marrow suppression occurred in patients receiving higher doses, but no clinical problems were seen except in 2 patients who required packed cell transfusion similar to their prior transfusions. CONCLUSION: (186)Re-HEDP is an effective radiopharmaceutical for the palliative treatment of metastatic bone pain and has minimal adverse effects.