Mouse monoclonal antibodies to transient receptor potential ankyrin 1 act as antagonists of multiple modes of channel activation.

The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in different pathophysiologies that include asthma, cough, itch, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and pain behaviors in rodents. Hence, TRPA1 antagonists are being pursued as potential therapeutics. With the goal of generating monoclonal antibodies (mAbs) to human TRPA1 that could act as selective antagonists, we immunized mice with a variety of antigens expressing TRPA1 channels. After generation of hybridomas, the hybridoma conditioned media were screened to identify the mAbs that bind TRPA1 channels by a flow cytometry assay utilizing U2OS or Chinese hamster ovary (CHO) cells stably expressing TRPA1. The purified IgGs from the hybridomas that showed selective binding to TRPA1 were evaluated for antagonism in agonist-induced (45)Ca(2+) uptake assays using CHO-TRPA1 cells. Several of the mAbs showed concentration-dependent inhibition of AITC and cold (4°C) activation of TRPA1. The most potent mAb, 2B10, had IC50 values of approximately 260 and 90 nM in the two assays, respectively. These antagonist mAbs also blocked osmotically activated TRPA1 as well as activation by an endogenous agonist (4-oxo-2-nonenal). In summary, we generated mouse mAbs against TRPA1 that act as antagonists of multiple modes of TRPA1 activation.

Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia.

Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.

Creation of a High-Yield AAV Vector Production Platform in Suspension Cells Using a Design-of-Experiment Approach.

Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, but vector manufacturing remains a challenge. New methods are needed to increase rAAV yields and reduce costs. Past efforts to improve rAAV production have focused on optimizing a single variable at a time, but this approach does not account for the interactions of multiple factors that contribute to vector generation. Here, we utilized a design-of-experiment (DOE) methodology to optimize rAAV production in a HEK293T suspension cell system. We simultaneously varied the transgene, packaging, and helper plasmid ratios, the total DNA concentration, and the cell density to systematically evaluate the impact of each variable across 52 conditions. The results revealed a unique set of parameters with a lower concentration of transgene plasmid, a higher concentration of packaging plasmid, and a higher cell density than previously described protocols. Using this DOE-optimized protocol, we achieved unpurified yields approaching 3 × 1014 viral genomes (VGs)/L of cell culture. Additionally, we incorporated polyethylene glycol (PEG)-based virus precipitation, pH-mediated protein removal, and affinity chromatography to our downstream processing, enabling average purified yields of >1 × 1014 VGs/L for rAAV-EGFPs across 13 serotypes and capsid variants.

Optimization and utilization of the SureFire phospho-STAT5 assay for a cell-based screening campaign.

The family of signal transducers and activators of transcription (STATs) consists of seven transcription factors that respond to a variety of cytokines, hormones, and growth factors. STATs are activated by tyrosine phosphorylation, which results in their dimerization and translocation into the nucleus where they exert their effect on transcription of regulated target genes. The phosphorylation of STATs is mediated mainly by Janus kinases (JAKs). The JAK/STAT pathway plays a critical role in hematopoietic and immune cell function. Here we focus on one member of the STAT family, STAT5. STAT5 is phosphorylated by several JAKs, including Jak3, Jak2, and Tyk2, in response to interleukin-2, erythropoietin (EPO), and interleukin-22, respectively. Activation of STAT5 is essential to T cell development and has been associated with hematologic malignancies. Therefore, the ability to assess STAT5 phosphorylation is important for discovery efforts targeting these indications. The assay formats available to detect phosphorylated STAT5 (pSTAT5) are relatively low throughput and involve lengthy protocols. These formats include western blot analysis, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The SureFire (Perkin Elmer, Waltham, MA) pSTAT5 assay is a homogeneous assay that utilizes AlphaScreen (Perkin Elmer) technology to detect pSTAT5 in cell lysates. We have used this assay format to evaluate EPO-induced STAT5 phosphorylation in HEL cells and successfully complete a small-scale screening campaign to identify inhibitors of this event. The results obtained in these studies demonstrate that the SureFire pSTAT5 assay is a robust, reliable assay format that is amenable to high-throughput screening (HTS) applications.

A signaling-enhanced chimeric receptor to activate the ICOS pathway in T cells.

Activation of the inducible costimulator (ICOS) signaling pathway in T cells is difficult to assess with bioassays, because most T cell lines do not constitutively express ICOS. Additionally, engagement of ICOS by its natural ligand B7 related protein 1 (B7RP1) is insufficient to elicit ICOS signaling, but requires simultaneous costimulation of the T cell receptor (TCR) to be effective. Here we describe a genetically engineered human T cell line that expresses a chimeric receptor (ICOS-CD3) consisting of full-length human ICOS fused at its C-terminal end to the cytoplasmic domain of human CD3 zeta. When engaged by B7RP1, ICOS-CD3 initiated signaling independently of TCR costimulation and induced substantially more IL-2 secretion in Jurkat T cells compared to wildtype ICOS. We demonstrate that this signaling-enhanced chimeric receptor can be used in simple and sensitive bioassays to detect bioactive B7RP1, anti-B7RP1 drugs, and the presence of corresponding neutralizing anti-drug antibodies.

Molecular mechanism of hepcidin-mediated ferroportin internalization requires ferroportin lysines, not tyrosines or JAK-STAT.

Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. In vitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. In vivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.

STK33 kinase activity is nonessential in KRAS-dependent cancer cells.

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors.

Application of lentiviral vectors for development of production cell lines and safety testing of lentiviral-derived cells or products.

Lentiviral vectors (LVs) are frequently used to engineer cell lines for preclinical research purposes including assay development and target validation. Development of production cell lines for manufacturing recombinant protein therapeutics may also benefit from the use of LVs because they may reduce timelines and generate more uniform or higher expressing stable pools and clones. In addition, LVs could be advantageous for engineering new, alternative host cell substrates due to their ability to efficiently transduce most cell types. We demonstrate here that NS0 mouse myeloma cells, a host cell frequently used for protein production, can be transduced with LVs to greater than 80% efficiency and with no cytotoxic effects. The use of LVs for engineering of production cell lines will require additional testing procedures. Since LVs have previously been used in human gene therapy clinical trials, safety testing assays and procedures have been developed that could easily be applied to the development process for manufacturing cell lines to ensure the absence of unwanted viral material in cell banks and biologic products.

Activity of panitumumab alone or with chemotherapy in non-small cell lung carcinoma cell lines expressing mutant epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) kinase domain mutations cause hyperresponsiveness to ligand and hypersensitivity to small-molecule tyrosine kinase inhibitors. However, little is known about how these mutations respond to antibodies against EGFR. We investigated the activity of panitumumab, a fully human anti-EGFR monoclonal antibody, in vitro in mutant EGFR-expressing non-small cell lung carcinoma (NSCLC) cells and in vivo with chemotherapy in xenograft models. Mutant EGFR-expressing NSCLC cells (NCI-H1975 [L858R+T790M] and NCI-H1650 [Delta746-750]) and CHO cells were treated with panitumumab before EGF stimulation to assess the inhibition of EGFR autophosphorylation. Established tumors were treated with panitumumab (25, 100, or 500 mug/mouse twice a week) alone or with docetaxel (10 or 20 mg/kg once a week) or cisplatin (7.5 mg/kg once a week). Antitumor activity and levels of proliferation markers were analyzed. Treatment of mutant EGFR-expressing CHO and NSCLC cells with panitumumab inhibited ligand-dependent autophosphorylation. In NCI-H1975 and NCI-H1650 xenografts, treatment with panitumumab alone or with cisplatin inhibited tumor growth compared with control (P < 0.0003). With panitumumab plus docetaxel, enhanced antitumor activity was seen in both xenografts versus panitumumab alone. Panitumumab treatment alone decreased Ki-67 and phospho- mitogen-activated protein kinase (pMAPK) staining in both xenografts compared with control. Docetaxel enhanced panitumumab activity in NCI-H1650 xenografts (decreased Ki-67 and pMAPK staining by >60%) when compared with either agent alone. Panitumumab inhibits ligand-induced EGFR phosphorylation, tumor growth, and markers of proliferation alone or with docetaxel in NSCLC cell lines that express clinically observed EGFR kinase domain mutations, including the small-molecule tyrosine kinase inhibitor-resistant T790M mutation.