Jiannan Feng

The N- and C-terminal carbohydrate recognition domains of galectin-9 contribute differently to its multiple functions in innate immunity and adaptive immunity

By binding to T cell Ig mucin-3 (Tim-3) expressed on different cells, galectin-9 (Gal-9) mediates two important functions, triggering T cell death and activating innate immune cells. The mechanisms by which ligation of the same molecule on different cell types mediates different effects are largely unclear. Gal-9 contains two carbohydrate recognition domains (CRD) in the N- and C-terminal regions (Gal-9-N and Gal-9-C). The N and C terminals of Gal-9 have been shown to have different activities in promoting T cell death. However, whether the differences between two domains account for its dual functions remains to be elucidated. Here we hypothesized that the different functions of Gal-9 in innate immunity and adaptive immunity are mediated by different domains. To test this, we created recombinant Gal-9 (Gal-9-NC) and homodimers containing either the NCRD (Gal-9-N) or the CCRD (Gal-9-C). All these Gal-9 constructs can activate dendritic cells (DCs) and induce T cell death. However, the Gal-9-C was much more potent than the Gal-9-N in inducing T cell death, while the Gal-9-N was much more effective in activating DCs by inducing much higher TNF-α and IL-6 production, greater phosphorylation of p38 and AKT. In both DC and T cells, Gal-9-N but not Gal-9-C stimulation resulted in markedly iκBα degradation. Finally, computer analyses suggested different patterns and affinities for the binding of the Gal-9-N and Gal-9-C to their receptor, Tim-3. Our data suggest that the N- and C-terminal CRDs of Gal-9 contribute differently to its ability to induce T cell death and to activate DCs. Further investigations on the underlying mechanisms will provide new insights into the biochemical basis for the multiple activities of Gal-9.

Receptor for activated C kinase 1 promotes hepatocellular carcinoma growth by enhancing mitogen‐activated protein kinase kinase 7 activity

c‐Jun N‐terminal protein kinase (JNK) is a member of the mitogen‐activated protein kinase (MAPK) superfamily. The activation of JNK is mediated by sequential protein phosphorylation through a MAPK module, namely, MAPK kinase kinase (MAP3K or MEKK) → MAPK kinase (MAP2K or MKK) → MAPK. Elevated levels of JNK activity have been frequently observed in hepatocellular carcinoma (HCC) and have been demonstrated to contribute to HCC growth by promoting HCC cell proliferation and resistance to tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL)‐ or Fas‐mediated apoptosis. Chronic inflammation contributes to the up‐regulation of JNK activity in HCC. However, it remains unknown whether aberrant JNK activity also results from some cell intrinsic defect(s). Here, we show that receptor for activated C kinase 1 (RACK1), an adaptor protein implicated in the regulation of multiple signaling pathways, could engage in a direct interaction with MKK7, the JNK‐specific MAP2K, in human HCC cells. Levels of RACK1 protein show correlation with the activity of the JNK pathway in human HCC tissues and cell lines. RACK1 loss‐of‐function or gain‐of‐function analyses indicate that RACK1 enhances MKK7/JNK activity in human HCC cells. Further exploration reveals that the interaction of RACK1 with MKK7 is required for the enhancement of MKK7/JNK activity by RACK1. RACK1/MKK7 interaction facilitates the association of MKK7 with MAP3Ks, thereby enhancing MKK7 activity and promoting in vitro HCC cell proliferation and resistance to TRAIL‐ or Fas‐mediated apoptosis as well as in vivo tumor growth. Conclusion: Overexpressed RACK1 augments JNK activity and thereby promotes HCC growth through directly binding to MKK7 and enhancing MKK7 activity. (HEPATOLOGY 2013)

BAFF-targeting therapy, a promising strategy for treating autoimmune diseases

Since B cell activating factor belonging to tumor necrosis factor (TNF) family (BAFF) has been identified as a critical factor for B cell maturation and survival, convincing evidence indicates that deregulation of BAFF is involved in pathogenesis of B cell related autoimmune diseases. Blockade of BAFF activity significantly improves the symptoms of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis both in animal models and clinical trials. Therefore, BAFF-targeting therapy is a promising approach to treat B cell related autoimmune diseases.

Two-mAb cocktail protects macaques against the Makona variant of Ebola virus.

A two–monoclonal antibody cocktail protects nonhuman primates against Ebola virus 3 days after lethal exposure. One-two punch for Ebola Antibody cocktails are an appealing therapeutic option for emerging infections such as the recent Ebola virus outbreak in West Africa because of their scalability and specificity. Qiu et al. report that the antibody cocktail used in Ebola virus–infected patients can be further simplified to only two antibodies and that these antibodies can be produced in engineered Chinese hamster ovary cells. This cocktail protected nonhuman primates against the virus responsible for the 2014–2015 outbreak up to 3 days after exposure. Combining these antibodies with those specific for other strains may lead to a broad ebolavirus therapy. The 2014–2015 Ebola virus (EBOV) outbreak in West Africa highlighted the urgent need for specific therapeutic interventions for infected patients. The human-mouse chimeric monoclonal antibody (mAb) cocktail ZMapp, previously shown to be efficacious in EBOV (variant Kikwit) lethally infected nonhuman primates (NHPs) when administration was initiated up to 5 days, was used in some patients during the outbreak. We show that a two-antibody cocktail, MIL77E, is fully protective in NHPs when administered at 50 mg/kg 3 days after challenge with a lethal dose of EBOV variant Makona, the virus responsible for the ongoing 2014–2015 outbreak, whereas a similar formulation of ZMapp protected two of three NHPs. The chimeric MIL77E mAb cocktail is produced in engineered Chinese hamster ovary cells and is based on mAbs c13C6 and c2G4 from ZMapp. The use of only two antibodies in MIL77E opens the door to a pan-ebolavirus cocktail.

Basal c-Jun NH2-terminal protein kinase activity is essential for survival and proliferation of T-cell acute lymphoblastic leukemia cells

Hyperactivation of c-Jun NH2-terminal protein kinase (JNK) has been found in various malignant lymphocytes and inhibition of JNK activity leads to cell cycle arrest and apoptosis. However, the role of JNK activity in the oncogenic growth of T-cell acute lymphoblastic leukemia (T-ALL) cells remains largely unknown. Here, we report that treatment of T-ALL cells with JNK inhibitors led to cell cycle arrest and apoptosis and increased sensitivity to Fas-mediated apoptosis, whereas weak ectopic expression of MKK7-JNK1 fusion protein, which shows constitutive JNK activity, in T-ALL cells resulted in accelerated cell cycle progression and resistance to Fas-mediated apoptosis. The protein levels of c-Myc and Bcl-2 were reduced in the presence of JNK inhibitors but were enhanced with MKK7-JNK1. Small interfering RNA against JNK1, but not JNK2, exhibited similar effects to JNK inhibitors. These findings suggest that targeting JNK, especially JNK1 isoform, may have some important therapeutic implications in the treatment of T-ALL. Further exploration revealed that JNK protein and basal JNK activity in T-ALL cells showed aberrant subcellular localization, but no hyperactivation of JNK was observed. Thus, our work suggests that there might be novel mechanism(s) other than hyperactivation underlying the protumorigenic role of JNK activity. [Mol Cancer Ther 2009;8(12):3214–22]

Novel chimeric anti-ricin antibody C4C13 with neutralizing activity against ricin toxicity

So far, no specific therapeutic agent is available for the treatment of ricin intoxication. Here, VH and VL genes were cloned from a hybridoma cell line secreting anti-ricin mAb 4C13, which could neutralize the toxicity of ricin. A chimeric antibody, c4C13, containing 4C13 mAb variable region genes fused to human constant region genes (gamma 1, kappa), was constructed. C4C13 retained the binding activity and recognized the same, or a closely related, epitope as the original mouse antibody. Furthermore, c4C13 blocked ricin-induced cytotoxicity to SP2/0 cells. Compared with its parental mouse antibody, c4C13 will be safer when used in human body to reverse clinical ricin intoxication.

Molecular Modeling and Affinity Determination of scFv Antibody: Proper Linker Peptide Enhances Its Activity

One of existing strategies to engineer active antibody is to link VH and VL domains via a linker peptide. How the composition, length, and conformation of the linker affect antibody activity, however, remains poorly understood. In this study, a dual approach that coordinates molecule modeling, biological measurements, and affinity evaluation was developed to quantify the binding activity of a novel stable miniaturized anti-CD20 antibody or single-chain fragment variable (scFv) with a linker peptide. Upon computer-guided homology modeling, distance geometry analysis, and molecular superimposition and optimization, three new linker peptides PT1, PT2, and PT3 with respective 7, 10, and 15 residues were proposed and three engineered antibodies were then constructed by linking the cloned VH and VL domains and fusing to a derivative of human IgG1. The binding stability and activity of scFv-Fc chimera to CD20 antigen was quantified using a micropipette adhesion frequency assay and a Scatchard analysis. Our data indicated that the binding affinity was similar for the chimera with PT2 or PT3 and ~24-fold higher than that for the chimera with PT1, supporting theoretical predictions in molecular modeling. These results further the understanding in the impact of linker peptide on antibody structure and activity.

IGF-1R and ErbB3/HER3 contribute to enhanced proliferation and carcinogenesis in trastuzumab-resistant ovarian cancer model

Trastuzumab (Herceptin®) has demonstrated clinical potential in several types of HER2-overexpressing human cancers. However, primary and acquired resistance occurs in many HER2-positive patients with regimens. To investigate the possible mechanism of acquired therapeutic resistance to trastuzumab, we have developed a preclinical model of human ovarian cancer cells, SKOV3/T, with the distinctive feature of stronger carcinogenesis. The differences in gene expression between parental and the resistant cells were explored by microarray analysis, of which IGF-1R and HER3 were detected to be key molecules in action. Their correctness was validated by follow-up experiments of RT-PCR, shRNA-mediated knockdown, downstream signal activation, cell cycle distribution and survival. These results suggest that IGF-1R and HER3 differentially regulate trastuzumab resistance and could be promising targets for trastuzumab therapy in ovarian cancer.

Disruption of TAB1/p38α Interaction Using a Cell-permeable Peptide Limits Myocardial Ischemia/Reperfusion Injury

Targeting the adaptor protein (transforming growth factor-β (TGF-β)-activated protein kinase 1 (TAK1)-binding protein 1) (TAB1)-mediated non-canonical activation of p38α to limit ischemia/reperfusion (I/R) injury after an acute myocardial infarction seems to be attractive since TAB1/p38α interaction occurs specifically in very limited circumstances and possesses unique structural basis. However, so far no TAB1/p38α interaction inhibitor has been reported due to the limited knowledge about the interfaces. In this study, we sought to identify key amino acids essential for the unique mode of interaction with computer-guided molecular simulations and molecular docking. After validation of the predicted three-dimensional (3-D) structure of TAB1/p38α complex, we designed several peptides and evaluated whether they could block TAB1/p38α interaction with selectivity. We found that a cell-permeable peptide worked as a selective TAB1/p38α interaction inhibitor and decreased myocardial I/R injury. To our knowledge, this is the first TAB1/p38α interaction inhibitor.

In vivo identification of the interaction site of ErbB2 extracellular domain with its autoinhibitor.

Direct interference with the transforming potential of ErbB2 has become a subject of great interest. Disruption of critical ErbB2 ectodomain interactions may lead to novel therapeutic approaches for the treatment of various tumors. The ErbB receptor signaling can be inhibited by rationally designed peptide mimetics based on the subdomains of ErbB ectodomain. The mimetics can bind to the ErbB receptor specifically and block inter‐receptor interactions, resulting in the growth inhibition of ErbB2‐overexpressing cells in vitro. In this study, three‐dimensional structure of herstatin, an autoinhibitor of ErbB2 and ErbB2 ectodomain complex was constructed by computer‐aided molecular modeling. The binding site on ErbB2 ectodomain for herstatin was determined at S1 domain. The mutants of ErbB2 ectodomain were constructed. The interactions of ErbB2 ectodomain and its mutants with herstatin were analyzed for the first time in living cells that coexpressed herstatin and ErbB2 ectodomain or the mutants. The S1 domain in ErbB2 ectodomain was verified as the interaction site with herstatin by immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer (FRET). The binding region of herstatin on ErbB2 ectodomain might be a potential target region for the drug design.