Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin.

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes.

Development of a novel immunoassay specific for mouse intact proinsulin.

The blood concentration of intact proinsulin, but not total proinsulin, has been suggested to be a diagnostic marker for type 2 diabetes mellitus (T2DM), but a sensitive assay specific for rodent intact proinsulin is lacking. Here, a novel enzyme-linked immunosorbent assay (ELISA) for mouse intact proinsulin was developed. The developed ELISA detected mouse intact proinsulin with the working range of 8.3 to 2700pg/ml. Cross-reactivity with mouse split-32,33 proinsulin was approximately 100times lower than the reactivity with mouse intact proinsulin, and no cross-reactivity with mouse insulin was detected. The developed ELISA was sufficiently sensitive to detect low levels of intact proinsulin in normal mouse plasma. The measurement by the developed ELISA revealed that intact proinsulin was elevated in the plasma of type 2 diabetic db/db mice as mice aged, and the ratio of intact proinsulin/insulin in plasma was correlated with levels of glycated hemoglobin A1c as seen in T2DM patients. These results suggest that the plasma level of intact proinsulin, but not total proinsulin, is a sensitive marker for pancreatic dysfunction and the ensuring diabetic disease progression of db/db mice. This ELISA could aid nonclinical evaluation of therapeutic interventions in T2DM.

Limited expression of reticulocalbin-1 in lymphatic endothelial cells in lung tumor but not in normal lung.

Lymphatic endothelial cells in tumors (T-LECs) are considered to have different characteristics from LECs in non-tumor tissues (N-LECs). However, differences between the two types have not been well analyzed at molecular level. In this report, we performed differential proteome analysis of T-LEC and N-LEC models prepared by cultivation of LECs in tumor conditioned medium. By expression profiling of identified proteins using tissue microarrays, reticulocalbin-1 was found to be expressed in clinical specimen-derived T-LECs and lung cancer cells but not N-LECs. It is suggested that reticulocalbin-1 may be an important molecule in understanding T-LEC function and control of lymphatic metastasis.

Selective blockade of transient receptor potential vanilloid 4 reduces cyclophosphamide-induced bladder pain in mice.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel activated by various physical stimuli such as cell swelling and shear stress. TRPV4 is expressed in bladder sensory nerves and epithelium, and its activation produces urinary dysfunction in rodents. However, there have been few reports regarding its involvement in bladder pain. Therefore, we investigated whether TRPV4 is involved in bladder pain in mouse cystitis model. Intraperitoneal injection of cyclophosphamide (CYP; 300 mg/kg) produced mechanical hypersensitivity in the lower abdomen associated with a severe inflammatory bladder in mice. The mechanical threshold was reversed significantly in Trpv4-knockout (KO) mice. Repeated injections of CYP (150 mg/kg) daily for 4 days provoked mild bladder inflammation and persistent mechanical hypersensitivity in mice. Trpv4-KO mice prevented a reduction of the mechanical threshold without an alteration in bladder inflammation. A selective TRPV4 antagonist also reversed the mechanical threshold in chronic cystitis mice. Although expression of Trpv4 was unchanged in the bladders of chronic cystitis mice, the level of phosphorylated TRPV4 was increased significantly. These results suggest involvement of TRPV4 in bladder pain of cystitis mice. A TRPV4 antagonist might be useful for patients with irritable bladder pain such as those with interstitial cystitis/painful bladder syndrome.

[From disease proteomics to biomarker development-establishment of antibody proteomics technology and exploration of cancer-related biomarkers].

Molecular biomarkers are keys to the development of new diagnostic protocols and therapies. Recently, significant research effort has been devoted to the development of these biomarkers using various approaches. Perhaps the most promising approach is disease proteomics. This method involves analyzing and identifying changes in the expression pattern at the protein level in the diseased condition (disease-related proteins) by using two-dimensional differential gel electrophoresis analysis (2D-DIGE). In the case of disease proteomics, hundreds of candidate disease-related proteins can be identified at a time. Therefore, how to pick the really valuable proteins up from a number of candidate drug targets is the most important issue to be solved worldwide. Here, we introduce a novel approach, termed "antibody proteomics", which addresses this issue. Using antibody proteomics it is possible to identify a variety of disease-related proteins by 2D-DIGE and simultaneously prepare monoclonal antibodies to these proteins by using a phage antibody library. The advantage of this technology is that the target proteins are identified in a high-throughput manner. Our approach relies on the fact that tissue microarray analysis can evaluate the relationship between disease-related proteins and disease progression, based on clinical and pathological information. In this review, we discussed the development and application of antibody proteomics and gave an overview of future work.

Sensitization of transient receptor potential vanilloid 4 and increasing its endogenous ligand 5,6-epoxyeicosatrienoic acid in rats with monoiodoacetate-induced osteoarthritis.

Transient receptor potential vanilloid 4 (TRPV4) receptor modulates pain, and this has been noted in several animal models. However, the involvement of TRPV4 in osteoarthritic (OA) pain remains poorly understood. This study assessed the functional changes in TRPV4 and the expression of its endogenous ligand 5,6-epoxyeicosatrienoic acid (5,6-EET) in a rat monoiodoacetate (MIA)-induced OA pain model (MIA rats). Monoiodoacetate-treated rats showed reduced grip strength as compared to sham-treated rats, and this loss in function could be recovered by the intraarticular administration of a TRPV4 antagonist (HC067047 or GSK2193874). By contrast, the intraarticular administration of the TRPV4 agonist, GSK1016790A, increased the pain-related behaviors in MIA rats but not in sham rats. TRPV4 expression was not increased in knee joints of MIA rats; however, the levels of phosphorylated TRPV4 at Ser824 were increased in dorsal root ganglion neurons. In addition, 5,6-EET was increased in lavage fluids from the knee joints of MIA rats and in meniscectomy-induced OA pain model rats. 5,6-EET and its metabolite were also detected in synovial fluids from patients with OA. In conclusion, TRPV4 was sensitized in the knee joints of MIA rats through phosphorylation in dorsal root ganglion neurons, along with an increase in the levels of its endogenous ligand 5,6-EET. The analgesic effects of the TRPV4 antagonist in the OA pain model rats suggest that TRPV4 may be a potent target for OA pain relief.

Improved cytosolic translocation and tumor-killing activity of Tat-shepherdin conjugates mediated by co-treatment with Tat-fused endosome-disruptive HA2 peptide.

Tat peptides are useful carriers for delivering biologic molecules into the cell for both functional analysis of intracellular disease-related proteins and treatment of refractory diseases. Most internalized Tat-fused cargos (Tat-cargos) are trapped within the endosome, however, which limits the biologic function of the cargo. In this study, we demonstrated that Tat-fused HA2 peptide (HA2Tat), an endosome disrupted peptide, enhanced the endosome-escape efficiency of Tat-cargos. In cells treated with a mixture of fluorescein isothiocyanate-labeled Tat and HA2Tat, widespread fluorescence was observed throughout the cytosol. In addition, this HA2Tat-mediated cytosolic delivery technique led to enhanced cytotoxicity of Tat-fused anti-cancer peptides, specifically shepherdin. Thus, we improved the function of the delivered molecules by co-treating with HA2Tat and propose that this is a useful method for the delivery of therapeutic macromolecules into the cytosol.

Identification and evaluation of metastasis-related proteins, oxysterol binding protein-like 5 and calumenin, in lung tumors.

Metastasis is an important prognosis factor in lung cancer, therefore, it is imperative to identify target molecules and elucidate molecular mechanism of metastasis for developing new therapeutics and diagnosis methods. We searched for metastasis-related proteins by utilizing a novel antibody proteome technology developed in our laboratory that facilitated efficient screening of useful target proteins. Two-dimensional differential in-gel electrophoresis (2D-DIGE) analysis identified sixteen proteins, which were highly expressed in metastatic lung cancer cells, as protein candidates. Monoclonal single-chain variable fragments (scFvs) binding to candidates were isolated from a scFv-displaying phage library by affinity selection. Tissue microarray analysis of scFvs binding to candidates revealed that oxysterol binding protein-like 5 (OSBPL5) and calumenin (CALU) were expressed at a significantly higher levels in the lung tissues of metastasis-positive cases than that in the metastasis-negative cases (OSBPL5; p=0.0156, CALU; p=0.0055). Furthermore, 80% of OSBPL5 and CALU double-positive cases were positive for lymph node metastasis. Consistent with these observations, overexpression of OSBPL5 and CALU promoted invasiveness of lung cancer cells. Conversely, knockdown of these proteins using respective siRNAs reversed the invasiveness of the lung cancer cells. Moreover, these proteins were expressed in lung tumor tissues, but not in normal lung tissues. In conclusion, OSBPL5 and CALU are related to metastatic potential of lung cancer cells, and they could be useful targets for cancer diagnosis and also for development of drugs against metastasis.

Development of an antibody proteomics system using a phage antibody library for efficient screening of biomarker proteins.

Proteomics-based analysis is currently the most promising approach for identifying biomarker proteins for use in drug development. However, many candidate biomarker proteins that are over- or under-expressed in diseased tissues are found by such a procedure. Thus, establishment of an efficient method for screening and validating the more valuable targets is urgently required. Here, we describe the development of an "antibody proteomics system" that facilitates the screening of biomarker proteins from many candidates by rapid preparation of cross-reacting antibodies using phage antibody library technology. Using two-dimensional differential in-gel electrophoresis analysis, 16 over-expressed proteins from breast cancer cells were identified. Specifically, proteins were recovered from the gel pieces and a portion of each sample was used for mass spectrometry analysis. The remainder was immobilized onto a nitrocellulose membrane for antibody-expressing phage enrichment and selection. Using this procedure, antibody-expressing phages against each protein were successfully isolated within two weeks. The expression profiles of the identified proteins were then acquired by immunostaining of breast tumor tissue microarrays with the antibody-expressing phages. Using this approach, expression of Eph receptor A10, TRAIL-R2 and Cytokeratin 8 in breast tumor tissues were successfully validated. These results demonstrate the antibody proteomics system is an efficient method for screening tumor-related biomarker proteins.

Creation and X-ray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor necrosis factor-alpha antagonist.

Tumor necrosis factor-alpha (TNF) induces inflammatory response predominantly through the TNF receptor-1 (TNFR1). Thus, blocking the binding of TNF to TNFR1 is an important strategy for the treatment of many inflammatory diseases, such as hepatitis and rheumatoid arthritis. In this study, we identified a TNFR1-selective antagonistic mutant TNF from a phage library displaying structural human TNF variants in which each one of the six amino acid residues at the receptor-binding site (amino acids at positions 84-89) was replaced with other amino acids. Consequently, a TNFR1-selective antagonistic mutant TNF (R1antTNF), containing mutations A84S, V85T, S86T, Y87H, Q88N, and T89Q, was isolated from the library. The R1antTNF did not activate TNFR1-mediated responses, although its affinity for the TNFR1 was almost similar to that of the human wild-type TNF (wtTNF). Additionally, the R1antTNF neutralized the TNFR1-mediated bioactivity of wtTNF without influencing its TNFR2-mediated bioactivity and inhibited hepatic injury in an experimental hepatitis model. To understand the mechanism underlying the antagonistic activity of R1antTNF, we analyzed this mutant using the surface plasmon resonance spectroscopy and x-ray crystallography. Kinetic association/dissociation parameters of the R1antTNF were higher than those of the wtTNF, indicating very fast bond dissociation. Furthermore, x-ray crystallographic analysis of R1antTNF suggested that the mutation Y87H changed the binding mode from the hydrophobic to the electrostatic interaction, which may be one of the reasons why R1antTNF behaved as an antagonist. Our studies demonstrate the feasibility of generating TNF receptor subtype-specific antagonist by extensive substitution of amino acids of the wild-type ligand protein.

Creation of novel cell-penetrating peptides for intracellular drug delivery using systematic phage display technology originated from Tat transduction domain.

Many biologically active proteins need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm. Cell penetrating peptides (CPPs) have been developed to efficiently deliver a wide variety of cargo in a fully biological active form into a range of cell types for the treatment of multiple preclinical disease models. To further develop this methodology, we established a systematic approach to identify novel CPPs using phage display technology. Firstly, we screened a phage peptide library for peptides that bound to the cell membrane. Secondly, to assess functionality as intracellular carriers, we recombined cDNAs of binding peptides with protein synthesis inhibitory factor (PSIF) to create fusion proteins. Randomly chosen clones were cultured and expression of peptide-PSIF fusion proteins induced, followed by screening of protein synthesis activity in cells. Using this systematic approach, novel and effective CPPs were rapidly identified. We suggest that these novel cell-penetrating peptides can utilized as drug delivery tools for protein therapy or an analytical tool to study mechanisms of protein transduction into the cytoplasm.

Creation of novel Protein Transduction Domain (PTD) mutants by a phage display-based high-throughput screening system.

Significant research effort is currently focused on Protein Transduction Domains (PTDs) as potential intracellular drug delivery carriers. However, the application of this technology is limited because the transduction efficiencies are often insufficient for therapeutic purposes, even using HIV-1 Tat peptide. Here we describe a high-throughput screening method based on a phage display system for isolating novel PTDs with improved cell penetration activity. The screening method involves using protein synthesis inhibitory factor (PSIF) as cargo of PTD. Using this method, several Tat-PTD mutants of superior cell-penetrating activity were isolated. Interestingly, the amino acid sequence of the PTD mutants contained some characteristic residues, such as proline. Thus, our screening method may prove useful in determining the relationship between protein transduction and amino acid sequence.

Quality enhancement of the non-immune phage scFv library to isolate effective antibodies.

The non-immune phage antibody library system is one of the most attractive technologies available to current therapeutic, diagnostic and basic scientific research. This system allows the rapid isolation of antibodies of interest that could subsequently be applied directly to drug delivery systems and antibody therapy. Previously, we reported the primer sets to encompass the antibody repertoire and thus improve library quality. However, a wide number of varying primer sets cause to decrease the amplification efficiency of antibody genes. In the present study, we re-generated the library primer sets newly and constructed an improved library from non-immune mice that was far superior in terms of variety and quality. This new library contained 2.4 billion independent clones. In addition, we optimized the selection step from this library to isolate high-affinity antibodies. The optimization of an affinity panning protocol by the incorporation of an automated Microfluidics instrument led to the successful isolation of three different monoclonal antibodies for human vascular endothelial growth factor receptor 2 (KDR). These antibodies were demonstrated to exhibit high specificity and were able to detect a mere 0.6 fmol of KDR by dot blot analysis. Previously reported antibodies for luciferase were also isolated successfully from this library. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies and the resulting isolation of antibodies for clinical and research applications.

A novel method for construction of gene fragment library to searching epitopes.

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.