Diabody-based recombinant formats of humanized IgG-like bispecific antibody with effective retargeting of lymphocytes to tumor cells

Recently, recombinant antibodies have been dissected into antigen-binding regions and rebuilt into multivalent high-avidity formats. These new structural designs are expected to improve in vivo pharmacokinetics and efficacy in clinical use. Here, we designed effective recombinant bispecific antibody (BsAb) formats based on hEx3, a humanized bispecific diabody with epidermal growth factor receptor and CD3 retargeting. The bispecific and bivalent IgG-like antibodies engineered from hEx3 (or its single-chain form, hEx3-scDb) and the human Fc region showed stronger binding to each target cell than did monovalent diabody formats, and their affinity was identical to that of the corresponding parent IgG. The bivalent effect of the constructed IgG-like BsAbs resulted in cell cytotoxicity 10 times that of monovalent diabodies, and further, the fusion of Fc portion contributed intense cytotoxicity in peripheral blood mononuclear cells by the induction of the antibody-dependent cellular cytotoxicity. The growth-inhibition effects of IgG-like BsAbs were superior to those of the approved therapeutic antibody cetuximab, which recognizes the same epidermal growth factor receptor antigen, even when peripheral blood mononuclear cells were used as effector cells. We thus demonstrated a critical improvement in the effect of hEx3 by the bottom-up construction of IgG-like BsAbs; in adoptive immunotherapy, monotherapy without supplemental molecules may be able to induce antibody-dependent cellular cytotoxicity.     

Antitumor activity of histone deacetylase inhibitors in non-small cell lung cancer cells: development of a molecular predictive model

To ascertain the potential for histone deacetylase (HDAC) inhibitor-based treatment in non-small cell lung cancer (NSCLC), we analyzed the antitumor effects of trichostatin A (TSA) and suberoylanilide hydroxamic acid (vorinostat) in a panel of 16 NSCLC cell lines via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TSA and vorinostat both displayed strong antitumor activities in 50% of NSCLC cell lines, suggesting the need for the use of predictive markers to select patients receiving this treatment. There was a strong correlation between the responsiveness to TSA and vorinostat (P < 0.0001). To identify a molecular model of sensitivity to HDAC inhibitor treatment in NSCLC, we conducted a gene expression profiling study using cDNA arrays on the same set of cell lines and related the cytotoxic activity of TSA to corresponding gene expression pattern using a modified National Cancer Institute program. In addition, pathway analysis was done with Pathway Architect software. We used nine genes, which were identified by gene-drug sensitivity correlation and pathway analysis, to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. The prediction performance of the support vector machine model was validated by an additional nine cell lines, resulting in a prediction value of 100% with respect to determining response to TSA and vorinostat. Our results suggested that (a) HDAC inhibitors may be promising anticancer drugs to NSCLC and (b) the nine-gene classifier is useful in predicting drug sensitivity to HDAC inhibitors and may contribute to achieving individualized therapy for NSCLC patients.     

Role of NADPH oxidase in tissue growth in a tissue engineering chamber in rats

Previously we described a subcutaneous arteriovenous loop (AVL)-based tissue engineering chamber system, which contains an intrinsic circulation circuit created by joining the proximal ends of the femoral artery and vein with a venous graft. We showed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was involved in mediating neovascularization inside the chamber. However, the role of NADPH oxidase in tissue formation in the chamber is unknown. In this study, we examined the effects of gp91ds-tat, a peptidyl inhibitor of NADPH oxidase, on the growth of engineered tissue blocks, using a rat chamber model. Chambers containing the AVL were filled with Matrigel mixed with gp91ds-tat (100 microM) or the scrambled control peptide. At 14 days, in control chambers, most of the Matrigel was replaced by granulation tissues; gp91ds-tat treatment significantly reduced the level of reactive oxygen species and retarded the tissue formation process. Although the total number of blood vessels per unit cellularized area was not different between two groups, most vessels in gp91ds-tat-treated tissues had smaller lumens as compared to control. The total area occupied by vessel lumens was much less in gp91ds-tat-treated tissues (10.3 +/- 1.3% in control vs. 1.7 +/- 0.5% in gp91ds-tat group; p < 0.001). In vitro, gp91ds-tat treatment reduced proliferation and migration of cultured microvascular endothelial cells. Our data suggest that inhibition of NADPH oxidase function retards tissue formation in the tissue engineering chamber, which may be related to the malformed new blood vessels in the engineered tissue.     

Effect of treatment with interferon α-2b and ribavirin in patients infected with genotype 2 hepatitis C virus

Aim:  Nearly 20% of chronic hepatitis C (CHC) patients with genotype 2 hepatitis C virus (HCV) infection are not curable, even by interferon (IFN)–ribavirin combination therapy. The aim of this study is to investigate the factors that determine the efficacy of combination therapy in patients with genotype 2 HCV infection.
Methods:  Fifty patients with CHC who underwent a treatment of 6 MU IFN α-2b with ribavirin for 24 weeks were retrospectively analyzed.
Results:  All the patients showed no serum HCV-RNA within 12 weeks after starting the therapy. Forty-one of the 50 patients (82%) achieved a sustained virological response (SVR). The age, sex, genotype (2a vs. 2b) and grade/stage of the liver by histopathology and pretreatment viral load werenot different between the sustained responders and relapsers. Univariate analysis showed that an earlier viral clearance from blood and a larger number of amino acid substitutions in the interferon sensitivity determining region (ISDR) were predictors of SVR. Multivariate analysis showed that a large number of amino acid substitutions in the ISDR was a predictor of SVR.
Conclusion:  The characterization of the amino acid sequences of ISDR may be helpful for predicting a relapse after combination therapy in patients with genotype 2 HCV infection.     

Differential superior prefrontal activity on initial versus subsequent shifts in naive subjects

Flexible adaptation to changing environments requires shifting of a cognitive set, one basic function of the prefrontal cortex. Set shifting, as instantiated in the Wisconsin Card Sorting Task (WCST) administered in a neuropsychological testing room, is typically achieved when subjects have no prior experiences of updating one WCST behavior to another. By contrast, earlier neuroimaging studies typically involved examination of repeated transitions between particular behaviors, to which situation subjects are far from naive. Naive subjects with no prior knowledge of the WCST were recruited in the present functional magnetic resonance imaging study to test set shifting under unknown situations that they experienced for the first time. Prominent activation was revealed in the left superior prefrontal cortex selectively on the initial shifts. On the other hand, the inferior prefrontal cortex was significantly activated on both the initial and subsequent shifts. The superior prefrontal activation distinguishable from the conventional inferior prefrontal activation suggests a selective role of this region in performance of the WCST in naive subjects.     

Long-Term Maintenance of Golimumab Effectiveness for Injection Spacing in Rheumatoid Arthritis Patients with Low Disease Activity Who Previously Received Other TNF Inhibitors: Minimum 2-year Data From an Observational Study

BackgroundGolimumab (GLM) has been reported to have lower immunogenicity than do other TNF inhibitors used for treating rheumatoid arthritis (RA). We previously found a prolonged effect of and improvement similar to that associated with infliximab (IFX) after switching to subcutaneous GLM (GLM-SC) for control of RA activity or adverse events. Thus, this study aimed to evaluate the continued maintenance of treatment efficacy and safety for > 2 years by switching to GLM-SC in RA patients with low disease activity or in remission after previous treatment with another tumor necrosis factor (TNF) inhibitor.MethodsThirty-two patients treated with etanercept or infliximab were switched to GLM-SC and maintained low disease activity. The patients were divided into two groups (GLMq4w and GLMq8w) through discussion with each patient, considering their general condition and convenience. The groups included patients with low disease activity or in remission who switched to 50-mg GLM therapy at 4-week and 8-week intervals, respectively.ResultsThe mean DAS28-ESR and DAS-CRP values in the GLMq4w group (17 patients) and GLMq8w group (15 patients) were maintained from baseline throughout the 104-week treatment period. Two patients from the GLMq4w group showed disease flaring to moderate disease activity. No serious adverse events occurred, and the treatment continuation rate at 104 weeks was 100% in both groups. After > 2 years of treatment, three patients in the GLMq8w group and one patient in the GLMq4w group discontinued GLM treatment due to relapse or complications. The 5-year survival rates were 88.2% and 75.5% in the GLMq4w and GLMq8w groups, respectively. The average treatment duration was 5.0 (2.0–7.5) years.ConclusionAdministration of GLM-SC at 4-week and 8-week intervals after switching from TNF inhibitors showed sustained long-term efficacy and acceptable safety in RA patients with low disease activity.     

Chronic buspirone treatment normalizes open field behavior in olfactory bulbectomized rats: assessment with a quantitative autoradiographic evaluation of the 5-HT1A binding sites

The olfactory bulbectomized (OBX) rat model of depression has been widely used in studies on the behavioral and neurochemical aspects of human depression. The objective of the present investigation was to assess open field (OF) activity and the brain regional 5-HT(1A) receptor densities of the sham operated (SHX) and OBX rats treated with saline (SHX-SAL, OBX-SAL), and either 10 mg/(kg day) (SHX-B10, OBX-B10) or 20 mg/(kg day) (SHX-B20, OBX-B20) of buspirone for 14 days, delivered by a subcutaneous osmotic minipump. Adult Sprague-Dawley rats were used for this experiment. The surgery was performed on the first day of the experiment and the rats were randomly assigned to either the SHX or OBX groups. The results of the OF tests were organized in eight groups. Following 14 days of treatment and the final OF tests, the rats were sacrificed and the brains were used for 5-HT(1A) receptor autoradiography using [(3)H]8-OH-DPAT. The data showed that the OF activities, 14 days following surgery, in the OBX rats were significantly elevated when compared to the SHX rats. In the OBX rats, only the 14-day treatment with 20mg/(kgday) of buspirone normalized the elevated OF activity, the same dose shown previously to be needed for the normalization of the regional 5-HT synthesis. A significant reduction in the number of 5-HT(1A) receptor sites was found in most brain regions in the OBX rats when compared to the SHX rats. Data also show that the regional density of the 5-HT(1A) receptors in OBX-SAL treated rats is lower than that of the SHX-SAL rats. The 14-day treatment with either 10 or 20 mg/(kg day) of buspirone reduced the 5-HT(1A) receptors in most brain regions of the SHX rats, without an obvious dose-dependent effect of the buspirone. The comparison between the OBX-B20 and control (SHX-B20) rats suggests that the buspirone treatment resulted in a regional balance in the 5-HT(1A) sites. A dose dependent reduction in the density of 5-HT(1A) sites was observed in the sham rats, but the buspirone treatment had very little effect on the density of the 5-HT(1A) receptors in the OBX rats. From these observations, we conclude that the antidepressant effects of buspirone in the OBX rat model of depression are likely mediated through the fine tuning of the regional imbalance of 5-HT(1A) receptors with even increases of about 20% in some limbic regions. The data suggest that the neurochemical effects of antidepressants should be studied in animal models of depression rather than in normal rats.     

Comparative study of the distribution of the alpha-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons

We compared the distribution of the α‐subunit mRNAs of voltage‐gated sodium channels Nav1.1–1.3 and Nav1.6–1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naïve DRG, the expression of Nav1.1 and Nav1.6 was restricted to A‐fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, ‐1.8, and ‐1.9 mRNAs were more abundant in C‐fiber neurons compared with A‐fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and ‐1.9 were preferentially expressed by TrkA neurons, other α‐subunits were expressed independently of TrkA expression. Actually, all IB4⁺ neurons expressed both Nav1.8 and ‐1.9, and relatively limited subpopulations of IB4⁺ neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naïve DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up‐regulated mainly in A‐fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell‐derived neurotrophic factor (GDNF) reversed this up‐regulation, the Nav1.3 induction was independent of either TrkA or GFRα1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply. J. Comp. Neurol. 510:188–206, 2008. © 2008 Wiley‐Liss, Inc.     

Diagnostic efficacy of free to total ratio of prostate-specific antigen and prostate-specific antigen velocity, singly and in combination, in detecting prostate cancer in patients with total serum prostate-specific antigen between 4 and 10 ng/ml

To assess the diagnostic efficacy of prostate-specific antigen (PSA)-related parameters, using a total of 226 patients with gray zone PSA who underwent prostate biopsy, various cutoff points of free to total ratio of PSA (f/t PSA) and PSA velocity (PSAV) were evaluated. Higher cutoff points of f/t PSA resulted in high sensitivity and negative predictive value (NPV): at f/t PSA <15%, sensitivity was 82.0% (41/50) and NPV 84.7% (50/59), and at f/t PSA <20%, 96.0% (48/50) and 92.3% (24/26). Lowering cutoff points also resulted in higher sensitivity and NPV: at PSAV ≥0.75 ng/ml per year, sensitivity was 71.4% (15/21) and NPV 82.4% (28/34), and at PSAV ≥0.40 ng/ml per year, 95.2% (20/21) and 95.2% (20/21). Further, among the patients with both of these parameters available, both sensitivity and NPV achieved 100% (10/10 and 7/7) when the indication for biopsy was determined as f/t PSA <15% or PSAV ≥0.40 ng/ml per year. Our results showed that unnecessary prostate biopsies could be more effectively avoided among patients with “gray zone PSA” by combination of f/t PSA and PSAV than single usage of these indexes.