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Antibody‐dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N‐Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8−/− Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)‐fucose synthesis, GDP mannose‐4,6‐dehydratase (Gmds) and GDP‐L‐fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX−/− and Gmds−/−, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of two proprietary IgG1 mAbs in the engineered host cells, and found that the titers were comparable to CHOZN® cells. The mAbs generated from either KO cell line exhibited loss of fucose modification, leading to significantly boosted FcγRIIIa binding and ADCC effects. Our data demonstrated that both FX−/− and Gmds−/− host cells could replace Fut8−/− CHO cells for clinical manufacturing of antibody therapeutics. [ABSTRACT FROM AUTHOR]

We demonstrate that c-Jun N-terminal kinase (JNK) responds to substrate stiffness and regulates adherens junction (AJ) formation in epithelial cells in 2D cultures and in 3D tissues in vitro and in vivo. Rigid substrates led to JNK activation and AJ disassembly, whereas soft matrices suppressed JNK activity leading to AJ formation. Expression of constitutively active JNK (MKK7-JNK1) induced AJ dissolution even on soft substrates, whereas JNK knockdown (using shJNK) induced AJ formation even on hard substrates. In human epidermis, basal cells expressed phosphorylated JNK but lacked AJ, whereas suprabasal keratinocytes contained strong AJ but lacked phosphorylated JNK. AJ formation was significantly impaired even in the upper suprabasal layers of bioengineered epidermis when prepared with stiffer scaffold or keratinocytes expressing MKK7-JNK1. By contrast, shJNK1 or shJNK2 epidermis exhibited strong AJ even in the basal layer. The results with bioengineered epidermis were in full agreement with the epidermis of jnk1-/- or jnk2-/- mice. In conclusion, we propose that JNK mediates the effects of substrate stiffness on AJ formation in 2D and 3D contexts in vitro as well as in vivo. [ABSTRACT FROM AUTHOR]

Although a lot of progress has been made in development of lentiviral vectors for gene therapy, the interactions of these vectors with cellular factors have not been explored adequately. Here we show that lentivirus infection phosphorylates JNK and that blocking the kinase activity of JNK decreases gene transfer in a dose-dependent manner, regardless of the viral envelope glycoprotein. Knockdown by small interfering RNA (siRNA) revealed that JNK1 but not JNK2 was required for productive gene transfer. The effect of JNK on gene transfer was not due to changes in the cell cycle, as JNK knockdown did not affect the cell cycle profile of target cells and even increased cell proliferation. In addition, confluent cell monolayers also exhibited JNK phosphorylation upon lentivirus infection and a dose-dependent decrease in gene transfer efficiency upon JNK inhibition. On the other hand, JNK activation was necessary for lentivirus internalization into the cell cytoplasm, while inhibition of JNK activity decreased virus entry without affecting binding to the cell surface. These experiments suggest that JNK is required for lentivirus entry into target cells and may have implications for gene transfer or for development of antiviral agents. [ABSTRACT FROM AUTHOR]

ABSTRACT We recently reported that c-Jun N-terminal kinase (JNK) is associated with adherens junctions and phosphorylates β-catenin at serine 33/37 and threonine 41. Here, we report that inhibition of JNK led to formation of adherens junctions, which was accompanied by dissociation of α-catenin from the β-catenin/ E-cadherin complex and increased association of α-catenin with the cytoskeleton. Conversely, activation of JNK increased binding of α-catenin to β-catenin, which was blocked by the JNK inhibitor SP600125 or JNK siRNA. In addition, inhibition of JNK failed to lead to adherens junction formation in cells where &alpha-catenin was absent or knocked down. Conversely, introduction of β-catenin restored the responsiveness of cells to JNK inhibition and led to cell-cell adhesion. Experiments with domain deletion mutants showed that binding of α-catenin to β-catenin was required for transport of adherens junction complexes to the cell surface, while binding to actin was required for translocation to the cell-cell contact sites. Collectively, our results suggest that JNK affects the association of α-catenin with the adherens junction complex and regulates adherens junctions. [ABSTRACT FROM AUTHOR]

Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8 -/- Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)-fucose synthesis, GDP mannose-4,6-dehydratase (Gmds) and GDP-L-fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX-/- and Gmds-/-, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of two proprietary IgG1 mAbs in the engineered host cells, and found that the titers were comparable to CHOZN® cells. The mAbs generated from either KO cell line exhibited loss of fucose modification, leading to significantly boosted FcγRIIIa binding and ADCC effects. Our data demonstrated that both FX-/- and Gmds-/- host cells could replace Fut8-/- CHO cells for clinical manufacturing of antibody therapeutics., (© 2020 Takeda Pharmaceutical Company Limited. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

We demonstrate that c-Jun N-terminal kinase (JNK) responds to substrate stiffness and regulates adherens junction (AJ) formation in epithelial cells in 2D cultures and in 3D tissues in vitro and in vivo. Rigid substrates led to JNK activation and AJ disassembly, whereas soft matrices suppressed JNK activity leading to AJ formation. Expression of constitutively active JNK (MKK7-JNK1) induced AJ dissolution even on soft substrates, whereas JNK knockdown (using shJNK) induced AJ formation even on hard substrates. In human epidermis, basal cells expressed phosphorylated JNK but lacked AJ, whereas suprabasal keratinocytes contained strong AJ but lacked phosphorylated JNK. AJ formation was significantly impaired even in the upper suprabasal layers of bioengineered epidermis when prepared with stiffer scaffold or keratinocytes expressing MKK7-JNK1. By contrast, shJNK1 or shJNK2 epidermis exhibited strong AJ even in the basal layer. The results with bioengineered epidermis were in full agreement with the epidermis of jnk1(-/-) or jnk2(-/-) mice. In conclusion, we propose that JNK mediates the effects of substrate stiffness on AJ formation in 2D and 3D contexts in vitro as well as in vivo.

Although a lot of progress has been made in development of lentiviral vectors for gene therapy, the interactions of these vectors with cellular factors have not been explored adequately. Here we show that lentivirus infection phosphorylates JNK and that blocking the kinase activity of JNK decreases gene transfer in a dose-dependent manner, regardless of the viral envelope glycoprotein. Knockdown by small interfering RNA (siRNA) revealed that JNK1 but not JNK2 was required for productive gene transfer. The effect of JNK on gene transfer was not due to changes in the cell cycle, as JNK knockdown did not affect the cell cycle profile of target cells and even increased cell proliferation. In addition, confluent cell monolayers also exhibited JNK phosphorylation upon lentivirus infection and a dose-dependent decrease in gene transfer efficiency upon JNK inhibition. On the other hand, JNK activation was necessary for lentivirus internalization into the cell cytoplasm, while inhibition of JNK activity decreased virus entry without affecting binding to the cell surface. These experiments suggest that JNK is required for lentivirus entry into target cells and may have implications for gene transfer or for development of antiviral agents.

We recently reported that c-Jun N-terminal kinase (JNK) is associated with adherens junctions and phosphorylates β-catenin at serine 33/37 and threonine 41. Here, we report that inhibition of JNK led to formation of adherens junctions, which was accompanied by dissociation of α-catenin from the β-catenin/E-cadherin complex and increased association of α-catenin with the cytoskeleton. Conversely, activation of JNK increased binding of α-catenin to β-catenin, which was blocked by the JNK inhibitor SP600125 or JNK siRNA. In addition, inhibition of JNK failed to lead to adherens junction formation in cells where α-catenin was absent or knocked down. Conversely, introduction of α-catenin restored the responsiveness of cells to JNK inhibition and led to cell-cell adhesion. Experiments with domain deletion mutants showed that binding of α-catenin to β-catenin was required for transport of adherens junction complexes to the cell surface, while binding to actin was required for translocation to the cell-cell contact sites. Collectively, our results suggest that JNK affects the association of α-catenin with the adherens junction complex and regulates adherens junctions.