NMR mapping of the highly flexible regions of 13C/15N-labeled antibody TTAC-0001-Fab.

Monoclonal antibody (mAb) drugs are clinically important for the treatment of various diseases. TTAC-0001 is under development as a new anti-cancer antibody drug targeting VEGFR-2. As the less severe toxicity of TTAC-0001 compared to Bevacizumab, likely due to the decreased in vivo half-life, seems to be related to its structural flexibility, it is important to map the exact flexible regions. Although the 13C/15N-labeled protein is required for NMR analyses, it is difficult to obtain antibody fragments (Fab and scFv) containing disulfide bonds through general cytosolic expression in Escherichia coli (E. coli). Here, we notably increased the periplasmic expression of the 13C/15N-labeled TTAC-0001-Fab (13C/15N-TTAC-Fab) through simple isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction at an increased optical density (1.5 OD600nm). Through NMR triple resonance experiments, two loop insertions (LI-1 between the VH and CH1; LI-2 between the VL and CL) were confirmed to be highly flexible. The additional LIs could be another way to engineer the antibody by changing the pharmacokinetic properties.

Phase I trial and pharmacokinetic study of tanibirumab, a fully human monoclonal antibody to vascular endothelial growth factor receptor 2, in patients with refractory solid tumors.

Background Tanibirumab is a fully human monoclonal antibody to vascular endothelial growth factor receptor 2 (VEGFR-2). We conducted a first-in-human phase I study of tanibirumab in patients with solid tumors refractory to standard chemotherapy. Primary endpoints were evaluating safety, pharmacokinetics (PKs), estimating maximum-tolerated dose (MTD) and recommended phase II dose (RP2D). Methods We designed our study to escalate tanibirumab at 9 different dose levels with a 3 + 3 method and tanibirumab (1-28 mg/kg) was administered intravenously on D1, 8, 15 in 28-day courses. Dose limiting toxicities (DLTs) were only assessed during the first cycle of treatment and response evaluation was performed every 2 cycles. The effects of tanibirumab on several angiogenic factors were analyzed. Results From October 2011 to September 2013, a total of 26 patients with refractory solid tumors were enrolled. The median age was 58 years (range, 27-75) and 20 patients were male. The most common tumor type was colorectal cancer (N = 19) and seven patients had a history of previous bevacizumab treatment. As hemangioma continued to occur, the final dose level, 28 mg/kg, was not performed. DLTs were not found, and the MTD was confirmed to be 24 mg/kg. Hemangioma was observed in 16 patients (61.5%), but all were grade 1-2 and disappeared after discontinuation of the study drug. Among the 18 patients in the efficacy set, no objective response was observed, but 11 patients showed stable disease. PKs were characterized by dose-dependent linear exposure and the mean trough concentrations exceeded biologically relevant target levels at 12 mg/kg and above. Serum VEGF, soluble VEGFR-2, and PlGF increased at the 4 mg/kg dose level and above. Conclusions Treatment with tanibirumab showed a tolerable toxicity profile and modest clinical efficacy in patients with refractory solid tumors. A phase II trial of tanibirumab is ongoing now.

A ligand-independent Tie2-activating antibody reduces vascular leakage in models of Clarkson disease.

Vascular dysfunction resulting from endothelial hyperpermeability is a common and important feature of critical illness due to sepsis, trauma, and other conditions associated with acute systemic inflammation. Clarkson disease [monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome (ISCLS)] is a rare, orphan disorder marked by spontaneous and recurrent episodes of hypotensive shock and peripheral edema due to widespread vascular leakage in peripheral tissues. Mortality from acute flares approaches 30% due to lack of effective therapies. We evaluated a monoclonal antibody (4E2) specific for the endothelial receptor tyrosine kinase Tie2 in ISCLS models. 4E2 activated Tie2 in ISCLS patient-derived endothelial cells and reduced baseline and proinflammatory mediator-induced barrier dysfunction. 4E2 also reduced mortality and/or vascular leakage associated with systemic histamine challenge or influenza infection in the SJL/J mouse model of ISCLS. These findings support a critical role for Tie2 dysregulation in ISCLS and highlight a viable therapeutic approach to this catastrophic disorder.

An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone.

Tumor progression is intimately associated with the vasculature, as tumor proliferation induces angiogenesis and tumor cells metastasize to distant organs via blood vessels. However, whether tumor invasion is associated with blood vessels remains unknown. As glioblastoma (GBM) is featured by aggressive invasion and vascular abnormalities, we characterized the onset of vascular remodeling in the diffuse tumor infiltrating zone by establishing new spontaneous GBM models with robust invasion capacity. Normal brain vessels underwent a gradual transition to severely impaired tumor vessels at the GBM periphery over several days. Increasing vasodilation from the tumor periphery to the tumor core was also found in human GBM. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) showed a spatial correlation with the extent of vascular abnormalities spanning the tumor-invading zone. Blockade of VEGFR2 suppressed vascular remodeling at the tumor periphery, confirming the role of VEGF-VEGFR2 signaling in the invasion-associated vascular transition. As angiopoietin-2 (ANGPT2) was expressed in only a portion of the central tumor vessels, we developed a ligand-independent tunica interna endothelial cell kinase 2 (Tie2)-activating antibody that can result in Tie2 phosphorylation in vivo. This agonistic anti-Tie2 antibody effectively normalized the vasculature in both the tumor periphery and tumor center, similar to the effects of VEGFR2 blockade. Mechanistically, this antibody-based Tie2 activation induced VE-PTP-mediated VEGFR2 dephosphorylation in vivo. Thus, our study reveals that the normal-to-tumor vascular transition is spatiotemporally associated with GBM invasion and may be controlled by Tie2 activation via a novel mechanism of action.

COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code.

Cystic fibrosis (CF) is a childhood hereditary disease in which the most common mutant form of the CF transmembrane conductance regulator (CFTR) DeltaF508 fails to exit the endoplasmic reticulum (ER). Export of wild-type CFTR from the ER requires the coat complex II (COPII) machinery, as it is sensitive to Sar1 mutants that disrupt normal coat assembly and disassembly. In contrast, COPII is not used to deliver CFTR to ER-associated degradation. We find that exit of wild-type CFTR from the ER is blocked by mutation of a consensus di-acidic ER exit motif present in the first nucleotide binding domain. Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24. We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in DeltaF508-expressing patients.

Preclinical pharmacokinetics, interspecies scaling, and pharmacokinetics of a Phase I clinical trial of TTAC-0001, a fully human monoclonal antibody against vascular endothelial growth factor 2.

BACKGROUND: VEGF is a highly selective mitogen that serves as the central regulator of tumor angiogenesis by mediating endothelial proliferation, permeability, and survival. Tanibirumab (TTAC-0001) is a fully human IgG1 monoclonal antibody derived from a fully human naïve single-chain variable fragment (ScFv) phage library that was developed to inhibit the effects of VEGF in the treatment of solid tumors, especially those of the brain. METHODS: In the present study, we conducted intravenous pharmacokinetic studies of TTAC-0001 in mice, rats, and cynomolgus monkeys. At the doses studied (3 mg/kg, 10 mg/kg, 30 mg/kg), TTAC-0001 exhibited dose proportionality in mice and monkeys. At a dose of ~10 mg/kg, the clearance of TTAC-0001 from serum was 0.017 mL/h in mice, 0.35 mL/h in rats, and 2.19 mL/h in cynomolgus monkeys, and the terminal half-life ranged from 20-30 h among the three species. Pharmacokinetic data in mice, rats, and cynomolgus monkeys were used to predict the pharmacokinetics of TTAC-0001 in humans using allometric scaling. The predicted serum clearance of TTAC-0001 in humans was 102.45 mL/h and the terminal half-life was 27.52 h. RESULTS: The maximum life span-corrected clearance value was 72.92 mL/h. The observed clearance in humans was more similar to the predicted scaled clearance. CONCLUSION: We investigated the pharmacokinetics of TTAC-0001 in mice, rats, and cynomolgus monkeys after intravenous administration. At the doses studied, TTAC-0001 exhibited dose proportionality in mice and monkeys. The scaled pharmacokinetics of TTAC-0001 reported here was useful for designing first-in-human studies. Allometric scaling in the therapeutic antibody is feasible.

Evaluation of drug mechanism and efficacy of a novel anti-angiogenic agent, TTAC-0001, using multi-modality bioimaging in a mouse breast cancer orthotopic model.

PURPOSE: Targeting of vascular endothelial growth factor receptors (VEGFRs) has potential anti-angiogenic effects because VEGFR-2 is the major signaling regulator of VEGF/VEGFR pathways. We aimed to elucidate the drug mechanism and anti-tumor efficacy of TTAC-0001, a novel, fully human anti-VEGFR-2/KDR monoclonal antibody, in mouse orthotopic breast cancer model using multi-modal bioimaging. MATERIALS AND METHODS: We used orthotopic xenograft tumor model in which human breast cancer cells (MDA-MB-231) were injected into the right mammary fat pad of Balb/c nude mice. We investigated its biodistribution using serial fluorescence imaging after injecting fluorescent-labelled-drug and mode of action using Matrigel plug angiogenesis assays. The anti-tumor efficacy of drug was assessed using ultrasonography and bioluminescence imaging. Histopathologic analyses, including hematoxylin and eosin staining and immunohistochemistry with anti-CD31 and anti-Ki-67 antibodies, were performed. Each experiment had four groups: control, bevacizumab 10 mg/kg (BVZ-10 group), TTAC-0001 2 mg/kg (TTAC-2 group), and TTAC-0001 10 mg/kg (TTAC-10 group). RESULTS: The TTAC-10 group showed good tumor targeting that lasted for at least 6 days and had a good anti-angiogenic effect with decreased hemoglobin content and fewer CD31-positive cells in the Matrigel plug. Compared with BVZ-10 and TTAC-2 groups, the TTAC-10 group showed the strongest anti-tumor efficacy, inhibiting tumor growth as detected by ultrasonography and bioluminescence imaging. The TTAC-10 group also showed the lowest viable tumor and micro-vessel areas and the lowest Ki-67 index in histopathologic analyses. CONCLUSION: We firstly demonstrated that TTAC-0001 effectively inhibited tumor growth and neovascularization in mouse orthotopic breast cancer model. It may provide a future treatment option for breast cancer.

Anticancer activity of TTAC-0001, a fully human anti-vascular endothelial growth factor receptor 2 (VEGFR-2/KDR) monoclonal antibody, is associated with inhibition of tumor angiogenesis.

Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.

TTAC-0001, a human monoclonal antibody targeting VEGFR-2/KDR, blocks tumor angiogenesis.

Angiogenesis is one of the most important processes for cancer cell survival, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and its receptor, particularly VEGF receptor-2 (VEGFR-2, or kinase insert domain-containing receptor, KDR), play critical roles in tumor-associated angiogenesis. We developed TTAC-0001, a human monoclonal antibody against VEGFR-2/KDR from a fully human naïve single-chain variable fragment phage library. TTAC-0001 was selected as a lead candidate based on its affinity, ligand binding inhibition and inhibition of VEGFR-2 signal in human umbilical vein endothelial cells (HUVEC). TTAC-0001 inhibited binding of VEGF-C and VEGF-D to VEGFR-2 in addition to VEGF-A. It binds on the N-terminal regions of domain 2 and domain 3 of VEGFR-2. It could inhibit the phosphorylation of VEGFR-2/KDR and ERK induced by VEGF in HUVEC. TTAC-0001 also inhibited VEGF-mediated endothelial cell proliferation, migration and tube formation in vitro, as well as ex vivo vessel sprouting from rat aortic rings and neovascularization in mouse matrigel model in vivo. Our data indicates that TTAC-0001 blocks the binding of VEGFs to VEGFR-2/KDR and inhibits VEGFR-induced signaling pathways and angiogenesis. Therefore, these data strongly support the further development of TTAC-0001 as an anti-cancer agent in the clinic.

Enhancement of B cell and monocyte populations in rats exposed to chlorpheniramine.

Chlorpheniramine is an anti-histamine agent on IgE-mediated inflammation. In order to investigate the immunomodulatory effects of chlorpheniramine, we assessed the changes of peripheral mononuclear cell populations and other general clinical parameters, including hematology and clinical chemistry, following chlorpheniramine administration in rats. Since prednisolone is commonly co-prescribed with anti-histamine in many hypersensitive reactions, we also examined the changes to compare the results after the prednisolone administration. Chlorpheniramine (50, 100 and 200 µg/kg) and prednisolone (1, 2 and 4 mg/kg) were intramuscularly administered to female Sprague-Dawley (SD) rats 3 times, at intervals of 1 week. Except the clinical signs, such as stiffness and abnormal gait due to the local toxicity at injection sites, no other significant changes in body weights, urinalysis, and macroscopic examination were noted in the animals given chlorpheniramine. On the other hand, white blood cells, especially B cells and monocytes, showed a dose-dependent increase in the chlorpheniramine-treated animals; whereas, the numbers of both B and T cells (helper T and cytotoxic T, NKT cells) were decreased in the prednisolone-treated animals. Taken together, these results suggest that chloropheniramine administration enhances white blood cells in the peripheral blood, mostly due to increases of the B cells and monocytes, but no T cells and NK cells.

Prognostic significance of c-Met expression in glioblastomas.

BACKGROUND: The authors investigated whether expression of c-Met protein in glioblastomas is associated with overall survival and biologic features representing tumor invasiveness in patients with glioblastomas. METHODS: Paraffin-embedded specimens of glioblastomas from 62 patients treated in a single institution were assessed by immunohistochemical (IHC) analysis of c-Met expression. On the basis of the clinical data for these patients, the association between c-Met expression and clinicobiologic features representing tumor invasiveness was analyzed. RESULTS: c-Met overexpression was detected in 29.0% (18 of 62) of glioblastomas. In patients with c-Met overexpression, the median survival was 11.7 months (95% confidence interval [95% CI], 9.9 months-13.5 months), compared with a median survival of 14.3 months (95% CI, 7.6 months-21.0 months) for patients whose tumors had no or little expression of c-Met (P=.031). On the radiographic analysis, 9 of 18 patients (50%) with tumors overexpressing c-Met demonstrated invasive and multifocal lesions on the initial magnetic resonance images, whereas only 9 of 44 patients (20.5%) with tumors that expressed no or little c-Met demonstrated these features (P=.030). Using immunohistochemistry, we also found a significant association between c-Met expression and matrix metalloproteinase-2,-9 (P=.020 and P=.013). Furthermore, Myc overexpression was found to be closely correlated with c-Met overexpression on IHC analysis (P=.004). CONCLUSIONS: The authors suggest that c-Met overexpression is associated with shorter survival time and poor treatment response in glioblastomas, the mechanism for which is elevated tumor invasiveness on the molecular and clinical phenotypes. This implies that more effective therapeutic strategies targeting c-Met receptors may have important clinical implication.

Improved sensitivity and physical properties of sol-gel protein chips using large-scale material screening and selection.

Protein chips are a powerful emerging technology with extensive biomedical applications. However, the development of optimal, economical surface materials capable of maintaining the activity of embedded proteins is a challenge. Here, we introduce a new optimized, low-cost, sol-gel biomaterial for use in protein chips with femtogram-level sensitivity. A novel protein chip material with significantly improved physical properties and sensitivity was produced using unique screening and selection methods. Using this platform, the sensitive, specific detection of the interactions between an HIV antigen and its antibody and between a cyclin-kinase protein pair was observed. This study is the first to demonstrate the detection of protein-protein interactions on sol-gel microarrays and describes an important improvement in the physical properties of sol-gel-derived protein chip materials for biomedical research.

ADP-ribosylation factor 4 small GTPase mediates epidermal growth factor receptor-dependent phospholipase D2 activation.

The epidermal growth factor receptor (EGFR) plays a critical role in the development, proliferation, and differentiation of cells of epithelial and mesenchymal origin. These EGFR-dependent cellular processes are mediated by a repertoire of intracellular signaling pathways triggered by the activation of the EGFR cytoplasmic domain, which originates from ligand binding of its extracellular domain. To understand the molecular mechanisms by which the intracellular domain of EGFR transmits mitogenic messages to the downstream signaling pathways, we used the cytoplasmic region of EGFR as bait in yeast two-hybrid screening. We found that ADP-ribosylation factor 4 (ARF4) interacts with the intracellular part of EGFR and mediates the EGF-dependent cellular activation of phospholipase D2 (PLD2) but does not mediate the activation of PLD1. In addition, ARF4-mediated PLD2 activation leads to dramatic activation of the transcription factor activator protein 1 (AP-1), and, importantly, ARF4 activity is required for EGF-induced activation of cellular AP-1. Our findings indicate that ARF4 is a critical molecule that directly regulates cellular PLD2 activity and that this ARF4-mediated PLD2 activation stimulates AP-1-dependent transcription in the EGF-induced cellular response.

Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway.

The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-DeltaF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000) Traffic 1, 852-870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNAp REceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells.