The novel DNA alkylating agent BO-1090 suppresses the growth of human oral cavity cancer in xenografted and orthotopic mouse models.

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 (γ-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.

Adenosine Deaminase 1 Overexpression Enhances the Antitumor Efficacy of Chimeric Antigen Receptor-Engineered T Cells.

Chimeric antigen receptor (CAR) T cell therapy mediates unprecedented benefit in certain leukemias and lymphomas, but has yet to achieve similar success in combating solid tumors. A substantial body of work indicates that the accumulation of adenosine in the solid tumor microenvironment (TME) plays a crucial role in abrogating immunotherapies. Adenosine deaminase 1 (ADA) catabolizes adenosine into inosine and is indispensable for a functional immune system. We have, for the first time, engineered CAR T cells to overexpress ADA. To potentially improve the pharmacokinetic profile of ADA, we have modified the overexpressed ADA in two ways, through the incorporation of a (1) albumin-binding domain or (2) collagen-binding domain. ADA and modified ADA were successfully expressed by CAR T cells and augmented CAR T cell exhaustion resistance. In a preclinical engineered ovarian carcinoma xenograft model, ADA and collagen-binding ADA overexpression significantly enhanced CAR T cell expansion, tumor tissue infiltration, tumor growth control, and overall survival, whereas albumin-binding ADA overexpression did not. Furthermore, in a syngeneic colon cancer solid tumor model, the overexpression of mouse ADA by cancer cells significantly reduced tumor burden and remodeled the TME to favor antitumor immunity. The overexpression of ADA for enhanced cell therapy is a safe, straightforward, reproducible genetic modification that can be utilized in current CAR T cell constructs to result in an armored CAR T product with superior therapeutic potential.

Expression analysis of an evolutionarily conserved metallophosphodiesterase gene, Mpped1, in the normal and beta-catenin-deficient malformed dorsal telencephalon.

We report the expression of the mouse Mpped1 in the telencephalon through embryonic stages to adulthood. Using Northern blotting analysis and RNA in situ hybridization (ISH), our data show that Mpped1 is specifically expressed in the brain and is enriched in the cortical plate of the developing telencephalon. Postnatally, the expression of Mpped1 is reduced in the cerebral cortex relative to its levels in the embryonic dorsal telencephalon. Also, Mpped1 expression is sustained in the hippocampal CA1 region. Examination of the expression of Mpped1 and other cortical layer markers by ISH in a malformed beta-catenin null dorsal telencephalon show that the Mpped1-, Cux2-, and Rorbeta-expressing superficial cortical layers are reduced and form patchy patterns, and the Tbr-1-expressing deep-layer neurons are incorrectly located on superficial layers, indicative of a migration defect of cortical neurons in the absence of beta-catenin.

Secretion of bispecific protein of anti-PD-1 fused with TGF-ß trap enhances antitumor efficacy of CAR-T cell therapy.

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 ß (TGF)-ß 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-ß, 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.

Enhanced insulin receptor interaction by a bifunctional insulin-transferrin fusion protein: an approach to overcome insulin resistance.

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.

Biodistribution, activation, and retention of proinsulin-transferrin fusion protein in the liver: Mechanism of liver-targeting as an insulin prodrug.

A recombinant proinsulin-transferrin fusion protein (ProINS-Tf) has been previously reported to be a novel long-lasting INS analog, acting specifically on the inhibition of hepatic glucose output. In this study, we investigated the biodistribution, activation and tissue retention of ProINS-Tf to elucidate its liver targeted anti-diabetic mechanism. The biodistribution study revealed that ProINS-Tf exhibited liver specific accumulation after a single intravenous injection, whereas transferrin (Tf) or insulin (INS) showed relatively even distribution among different organs. The conversion of inactive ProINS-Tf into an active immune-reactive INS-Tf form (irINS-Tf) via a Tf receptor (TfR) mediated process only occurred in the liver, but not in other organs. In addition, ProINS-Tf demonstrated a prolonged retention in the liver after an intravenous injection, suggesting the enhanced association of the bifunctional active form, irINS-Tf, within liver cells. Taken together, our results indicate that ProINS-Tf is a highly liver-targeted INS prodrug with a combination of 3 specific actions in liver cells: (1) TfR-mediated binding and uptake of the prodrug on the cell surface, (2) liver-specific, TfR-mediated conversion of the prodrug into its active form, and (3) the bifunctional binding of the active fusion protein to both Tf and INS receptors in the liver to achieve prolonged retention and thus enhanced anti-diabetic activities.

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc)2), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc)2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc)2-mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc)2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics.

DDX3 as a strongest prognosis marker and its downregulation promotes metastasis in colorectal cancer.

BACKGROUND: Conflicting results regarding the role of DEAD-box polypeptide 3 (DDX3) are seen not only between cancer types but also within the same type of cancer. In this study, we aimed at clarifying the prognostic significance of DDX3 in patients of major cancer types through large cohort survival analysis and further investigated its effects on cancer progression. METHODS: Large cohort survival analysis of 7 cancer types, including colorectal cancer, breast cancer, lung cancer, head and neck cancer, liver cancer, glioblastoma, and ovarian cancer, was performed using public database at RNA level and was further confirmed by IHC analysis at protein level. Phenotype parameters of DDX3 knockdown colon cancer cells and the mechanism of DDX3 regulated cancer progression were investigated in vitro and in vivo. RESULTS: In large cohort survival analysis, DDX3 had a significant prognostic predictive power in colorectal cancer at both RNA and protein level. Patients with low DDX3 expression had poor prognosis and frequent distant metastasis. Knockdown of DDX3 enhanced the migration and invasion abilities of colon cancer cells and promoted tumor metastasis in vivo. Snail upregulation with decreased membranous E-cadherin expression and reduced cell aggregation were found after DDX3 downregulation. CONCLUSIONS: Our study revealed the strong prognostic effect of DDX3 on colorectal cancer among seven major cancer types through larger cohort survival analysis at RNA and protein level. Low DDX3 expression promotes Snail/E-cadherin pathway mediated cancer metastasis and poor clinical outcome in colorectal cancer patients.