Despite the remarkable success of chimeric antigen receptor-modified T (CAR-T) cell therapy for blood malignancies, the clinical efficacy of this novel therapy in solid tumor treatment is largely limited by the immunosuppressive tumor microenvironment (TME). For instance, immune checkpoints (e.g., programmed cell death protein 1 [PD-1]/programmed death ligand 1 [PD-L1]) in TME play an important role in inhibiting T cell proliferation and functions. Transforming growth factor β (TGF)-β secreted by cancer cells in TME induces regulatory T cells (Tregs) and inhibits cytotoxic T cells. To overcome the inhibitory effect of immune checkpoints, we have previously engineered CAR-T cells to secrete anti-PD-1 to block the PD-1/PD-L1 pathway activity, a step demonstrating superior antitumor efficacy compared with conventional CAR-T cells. In this study, we engineered CAR-T cells that secrete bispecific trap protein co-targeting PD-1 and TGF-β, with the aim of further improving antitumor immunity. Compared with conventional CAR-T cells and anti-PD-1-secreting CAR-T cells, data from in vitro and in vivo experiments showed that CAR-T cells with trap protein secretion further attenuated inhibitory T cell signaling, enhanced T cell persistence and expansion, and improved effector function and resistance to exhaustion. In the xenograft mouse model, CAR-T cells with trap protein secretion exhibited significantly enhanced antitumor immunity and efficacy. With these observations, we demonstrate the potential of trap protein self-secreting CAR-T cells as a potent therapy for solid tumors.
Oral cancer is the fourth-most common cause of death in males and overall the sixth-most common cause of cancer death in Taiwan. Surgery, radiotherapy and chemotherapy combined with other therapies are the most common treatments for oral cavity cancer. Although cisplatin, 5-fluorouracil and docetaxel are commonly used clinically, there is no drug specific for oral cavity cancer. Here, we demonstrated that derivatives of 3a-aza-cyclopenta[a]indene, a class of newly synthesized alkylating agents, may be drugs more specific for oral cancer based on its potent in vitro cytotoxicity to oral cancer cells and on in vivo xenografts. Among them, BO-1090, bis(hydroxymethyl)-3a-aza-cyclopenta[a]indene derivative, targeted DNA for its cytotoxic effects as shown by inhibition of DNA synthesis (bromodeoxyuridine-based DNA synthesis assay), induction of DNA crosslinking (alkaline gel shift assay), and induction of DNA single-stranded breaks (Comet assay) and double-stranded breaks (c-H2AX focus formation). Following DNA damage, BO-1090 induced G1/S-phase arrest and apoptosis in oral cancer cell lines. The therapeutic potential of BO-1090 was tested in mice that received a xenograft of oral cavity cancer cell lines (SAS or Cal 27 cells). Intravenous injection of BO-1090 significantly suppressed tumor growth in comparison to control mice. BO-1090 also significantly reduced the tumor burden in orthotopic mouse models using SAS cells. There was no significant adverse effect of BO-1090 treatment with this dosage based on whole blood count, biochemical enzyme profiles in plasma and histopathology of various organs in mouse. Taken together, our current results demonstrate that B0-1090 may have potential as a treatment for oral cavity cancer.Oral cavity cancer involving the lesions occurred in the tongue, oropharynx and floor of the mouth is a prevalent type of cancer in developing countries such as India, Pakistan, Bangladesh and Taiwan. 1 In south-central Asia, it is the third-most common cancer, with an average incidence rate of 1 to 10 per 100,000 people. The incidence of oral cancer is also increasing in developed countries. 2 The treatments of oral cancer include surgery, radiation or chemotherapy, depending on the stage and nature of the tumor. 3,4 Unfortunately, only marginal improvement is seen in many patients, and a complete cure is often not achieved. Half of the patients diagnosed with oral cancer succumb to death within 5 years, and for those who survive, the quality of life remains poor. 1 Thus, development of a drug specific for oral cavity cancer is needed so that substantial improvement in treatment can be achieved.The cytotoxic and antitumor properties of alkylating agents are due to their ability to covalently bind to DNA. There are two types of DNA alkylating agents, monofunctional and bifunctional agents. Monofunctional alkylating agents mainly damage DNA by forming methylated adducts, whereas the damage induced by bifunctional alkylating agents includes monoadducts, intrastrand crosslinks and interstrand crossli...
Bifunctional fusion protein design has been widely utilized as a strategy to increase the efficacy of protein therapeutics. Previously, we proposed a novel application of the bifunctional fusion protein design through the introduction of proinsulin-transferrin (ProINS-Tf) fusion protein as a liver-specific protein prodrug to achieve a glucose-lowering effect in type 1 diabetic mice. In this report, we studied the binding characteristics of this activated fusion protein to the insulin receptor to elucidate its mechanism in eliciting insulin receptor-mediated signaling. We found that, with the assistance of the transferrin moiety binding to the transferrin receptor, the activated ProINS-Tf exhibited significantly higher binding affinity to the insulin receptor compared with the native insulin, resulting in a prolonged and stronger Akt phosphorylation. This enhanced induction by activated ProINS-Tf overcame insulin resistance in palmitate-treated HepG2 cells. ProINS-Tf also demonstrated a better glucose-lowering effect than native insulin, even with a much lower dose and less frequent injections, in non-obese diabetic mice with insulin resistance symptoms. The activated ProINS-Tf, serving as a bivalent protein molecule, could be a new insulin analog to overcome insulin resistance, which is associated with several diseases, including type 2 diabetes and non-alcoholic fatty liver disease. Insulin (INS) resistance is the major cause of the development of type 2 diabetes (T2D), and it is often referred as a state in which a higher than normal level of INS is required to achieve the normal response 1-3. INS resistance may result from alterations at different cellular levels, including insulin deficient signaling, inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction 3. Type 2 diabetic patients often require intensive insulin treatment to maintain glycemic control, which leads to increased risk of hypoglycemia, weight gain, and further deterioration of INS resistance state 1,4. Previously, INS X10 with higher IR binding affinity have been studied as a rapid-acting INS analogue to treat type 2 diabetes 5. INS X10 demonstrated sustained effect in inducing IR-mediated signaling; however, the development of INS X10 was discontinued due to disproportionate increase in mitogenic activity and higher breast cancer incidence in the long-term rat studies 6. It is still a great challenge to develop novel INS analogues with enhanced binding affinity to the IR to overcome INS resistance safely and effectively. A proinsulin-transferrin (ProINS-Tf) fusion protein was previously developed as a novel long-acting and liver-targeted INS prodrug for treating type 1 diabetic (T1D) mice 7,8. Proinsulin (ProINS), as a precursor of insulin (INS), has a much lower binding affinity to INS receptor (IR) and the resultant biologic potency is only 1% or less relative to INS 9. Therefore, ProINS-Tf is initially inactive and requires a lag time to be activated before exhibiting activity in stimulating Akt phosphorylation in H4IIE ce...
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