The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor. It heterodimerizes with aryl hydrocarbon nuclear translocator, binds to the xenobiotic-responsive element (XRE), and enhances the transcription of genes encoding xenobiotic metabolizing enzymes. AHR also plays important roles in the inhibition of intestinal carcinogenesis and the modulation of gut immunity. It is very important to screen for AHR-activating compounds because those are expected to produce the AHR-mediated physiological functions. Until now, AHR-mediated transcriptional activity represented by the transcriptional activity of CYP1A1 in luciferase assay has been applied as a screening procedure for AHR-activating compounds. However, the AHR-mediated transcriptional activity did not necessarily correspond with the CYP1A1 transcriptional activity. To evaluate AHRmediated transcriptional activity more specifically, and to screen for AHR-activating compounds, we establish a stable AHR-responsive HepG2 cell line by co-transfection of an AHR expression vector and an AHR-responsive vector (pGL3-XRE) containing a luciferase gene and three tandemly arranged XRE elements into a human hepatoma derived cell line, HepG2. The induction of luciferase activity in the stable AHR-responsive HepG2 cell line by typical AHR activators occurred in time-and concentrationdependent manners. By assessing the AHR target genes CYP1A1, UGT1A1, and ABCG2, an AHR activator-mediated induction was observed at mRNA level. Furthermore, the AHR activator-mediated induction of luciferase activity was positively correlated with the mRNA levels of CYP1A1, UGT1A1, and ABCG2. These findings verified the usefulness of the established stable AHR-responsive HepG2 cell line for the screening of AHR-activating compounds.

Xenobiotics are usually detoxified by drug-metabolizing enzymes and excreted from the body. The expression of many of drug-metabolizing enzymes is regulated by the aryl hydrocarbon receptor (AHR). Some substances in vegetables have the potential to be AHR ligands. To search for vegetable components that exhibit AHR-mediated transcriptional activity, we assessed the activity of vegetable extracts and identified the active compounds using the previously established stable AHR-responsive HepG2 cell line. Among the hot water extracts of vegetables, the highest activity was found in ginger. The ethyl acetate fraction of the ginger hot water extract remarkably induced AHR-mediated transcriptional activity, and the major active compound was found to be 6-shogaol. Subsequently, the mRNA levels of AHR-targeting drug-metabolizing enzymes (CYP1A1, UGT1A1, and ABCG 2) and the protein level of CYP1A1 in HepG2 cells were shown to be increased by 6-shogaol. This is the first report that 6-shogaol can regulate the expression of detoxification enzymes by AHR activation.

was determined using cell-based 4-1BB bioassay and co-culture assay with human peripheral blood mononuclear cells (hPBMC). Also, in vivo anti-tumor efficacy of YH32367 (ABL105) was assessed in both HER2-expressing tumor-bearing hPBMC humanized mice and h4-1BB knock-in mice.Results: A novel HER2/4-1BB BsAb, YH32367 simultaneously targets human epidermal growth factor receptor 2 (HER2) and h4-1BB and binds to both targets. YH32367 exhibited a strong 4-1BB signal activation in HER2-expressing tumor cells such as NCI-N87, JIMT-1 and HCC1954 cells. YH32367 stimulated IFN-g secretion and thereby inducing tumor cells lysis in co-culture assay with hPBMC and HER2-expressing tumor cells. YH32367 also showed superior efficacies on immune cells activation as well as tumor growth inhibition in both HER2-expressing tumor-bearing hPBMC humanized mice and h4-1BB knock-in mice models compared to the equimolar dosing of trastuzumab and benchmark 4-1BB agonistic antibodies. In particular, even a single dosing of YH32367 at doses of 1, 3 and 10 mg/kg elicited complete tumor regression in most tumors of MC38/hHER2-bearing h4-1BB knock-in mice. Peripheral T cell increase was not observed in these in vivo studies.