Vaccination with DKK1-derived peptides promotes bone formation and bone mass in an aged mouse osteoporosis model.

The investigation of agents for the treatment of osteoporosis has been a long-standing effort. The Wnt pathway plays an important role in bone formation and regeneration, and expression of Wnt pathway inhibitors, Dickkopf-1 (DKK1), appears to be associated with changes in bone mass. Inactivation of DKK1 leads to substantially increased bone mass in genetically manipulated animals. DKK1-derived peptides (DDPs) were added to BMP2-stimulated MC3T3-E1 preosteoblastic cells in vitro to evaluate inhibitory activity of DDPs in MC3T3-E1 cell differentiation. Study was extended in vivo on old female mice to show whether or not inhibition of endogenous DKK1 biological activity using DDPs vaccination approach leads to increase of bone formation, bone density, and improvement of bone microstructure. We reported that synthetic DDPs were able to reduce alkaline phosphatase activity, prevent mineralization and inhibit the differentiation of MC3T3-E1 cells in vitro. Furthermore, vaccination with these DDPs in aged female mice 4 times for a total period of 22 weeks promoted bone mass and bone microstructure. 3D microCT and histomorphometric analysis showed that there were significant increase in bone mineral densities, improvement of bone microstructure and promotion of bone formation in the vaccinated mice, especially in the mice vaccinated with DDP-A and DDP-C. Histological and scanning electron microscopy image analysis also indicated that vaccination increased trabecular bone mass and significantly decreased fragmentation of bone fibers. Taken together, these preclinical results suggest that vaccination with DDPs represents a promising new therapeutic approach for the treatment of bone-related disorders, such as osteoporosis.

IL-13 antibodies influence IL-13 clearance in humans by modulating scavenger activity of IL-13Rα2.

Human studies using Abs to two different, nonoverlapping epitopes of IL-13 suggested that epitope specificity can have a clinically significant impact on clearance of IL-13. We propose that Ab modulation of IL-13 interaction with IL-13Rα2 underlies this effect. Two Abs were administered to healthy subjects and mild asthmatics in separate dose-ranging studies and allergen-challenge studies. IMA-638 allows IL-13 interaction with IL-13Rα1 or IL-13Rα2 but blocks recruitment of IL-4Rα to the IL-13/IL-13Rα1 complex, whereas IMA-026 competes with IL-13 interaction with IL-13Rα1 and IL-13Rα2. We found ∼10-fold higher circulating titer of captured IL-13 in subjects treated with IMA-026 compared with those administered IMA-638. To understand how this difference could be related to epitope, we asked whether either Ab affects IL-13 internalization through cell surface IL-13Rα2. Humans inducibly express cell surface IL-13Rα2 but lack the soluble form that regulates IL-13 responses in mice. Cells with high IL-13Rα2 expression rapidly and efficiently depleted extracellular IL-13, and this activity persisted in the presence of IMA-638 but not IMA-026. The potency and efficiency of this clearance pathway suggest that cell surface IL-13Rα2 acts as a scavenger for IL-13. These findings could have important implications for the design and characterization of IL-13 antagonists.

Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides.

Th17 cells are a distinct lineage of effector CD4(+) T cells characterized by their production of interleukin (IL)-17. We demonstrate that Th17 cells also expressed IL-22, an IL-10 family member, at substantially higher amounts than T helper (Th)1 or Th2 cells. Similar to IL-17A, IL-22 expression was initiated by transforming growth factor beta signaling in the context of IL-6 and other proinflammatory cytokines. The subsequent expansion of IL-22-producing cells was dependent on IL-23. We further demonstrate that IL-22 was coexpressed in vitro and in vivo with both IL-17A and IL-17F. To study a functional relationship among these cytokines, we examined the expression of antimicrobial peptides by primary keratinocytes treated with combinations of IL-22, IL-17A, and IL-17F. IL-22 in conjunction with IL-17A or IL-17F synergistically induced the expression of beta-defensin 2 and S100A9 and additively enhanced the expression of S100A7 and S100A8. Collectively, we have identified IL-22 as a new cytokine expressed by Th17 cells that synergizes with IL-17A or IL-17F to regulate genes associated with skin innate immunity.

Complement C3a, CpG oligos, and DNA/C3a complex stimulate IFN-α production in a receptor for advanced glycation end product-dependent manner.

The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.

Vaccination with xenogeneic tumor endothelial proteins isolated in situ inhibits tumor angiogenesis and spontaneous metastasis.

Angiogenesis is critical for tumor growth and metastasis. Tumor tissues induce the expression of angiogenesis-associated proteins on endothelial surface that can be targeted for tumor immunotherapy. In our study, the rat tumor endothelial proteins (EP) were isolated in situ via biotinylation of tumor vascular endothelial luminal surface followed by streptavidin affinity chromatography. The isolated tumor EP contained numerous up-regulated angiogenesis-associated endothelial proteins. The administration of these tumor EP as a vaccine to mice reduced the microvessel density in subcutaneous primary LLC tumors, delayed spontaneous LLC tumor metastasis and prolonged post-surgery life span. T lymphocytes from tumor EP-vaccinated mice lysed human umbilical vascular endothelial cells, but not tumor cells in vitro, in a dose-dependent manner. Furthermore, adoptive transfer of antitumor EP antibodies in vivo targeted to tumor endothelium and inhibited spontaneous LLC tumor metastasis. This study provides a successful preclinical exploration of the active immunotherapy for tumor by targeting tumor angiogenesis.

Anti-apoptotic effects of a calpain inhibitor on cardiomyocytes in a canine rapid atrial fibrillation model.

PURPOSE: This study was designed to evaluate the effects of a calpain inhibitor on cardiac muscle apoptosis in rapid pacing canine atrial fibrillation (AF) models. METHODS: Twenty one dogs were divided into three groups: a sham operation group, a control AF group and a calpain inhibitor group. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute. N-Acetyl-Leu-Leu-Met (1.0 mg/kg/day) was administered in the calpain inhibitor group for three weeks. The activity of calpain I and cardiomyocyte apoptosis were measured by fluorometry and TUNEL assay, respectively. Protein expression of caspase-3 was detected by Western blot. The localizations of caspase-3, caspase-8, bcl-2 and ARC were assessed by immunohistochemistry. RESULTS: In comparison to the sham operation group, the activity of calpain I was significantly increased in the control AF group (2.3 fold, p < 0.001), and decreased in the calpain inhibitor group (1.1 fold, p < 0.005). The calpain activity correlated with the apoptosis index (r = 0.9, p < 0.05). The apoptosis index was 1.0 +/- 0.2%, 11.8 +/- 6.8% and 3.5 +/- 2.1% in the sham operation group, control AF group and calpain inhibitor group, respectively. In the sham operation group, control AF group and calpain inhibitor group, the expressions of caspase-3 (13.0 +/- 1.9%, 52.8 +/- 4.3% and 33.6 +/- 3.7%), caspase-8 (40.1 +/- 5.3%, 92.6 +/- 6.5% and 55.3 +/- 5.9%), bcl-2 (65.8 +/- 6.1%, 52.0 +/- 5.7% and 69.9 +/- 5.3%) and ARC (70.2 +/- 8.6%, 68.8 +/- 7.3% and 81.5 +/- 8.8%) were calculated as immunohistochemical indexes, respectively. CONCLUSIONS: The calpain inhibitor N-Acetyl-Leu-Leu-Met attenuated apoptosis through a complicated network of apoptosis-related proteins, which may result in improvement of structural remodeling in atrial fibrillation.

Interleukin-13 neutralization by two distinct receptor blocking mechanisms reduces immunoglobulin E responses and lung inflammation in cynomolgus monkeys.

Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Ralpha1/IL-4Ralpha) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Ralpha, and Ab02 blocks IL-13 interaction with IL-13Ralpha1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Ralpha-binding epitope or the IL-13Ralpha1-binding epitope.

Novel agonist monoclonal antibodies activate TrkB receptors and demonstrate potent neurotrophic activities.

Tyrosine kinase receptor B (TrkB) mediates neurotrophic effects of brain-derived neurotrophic factor (BDNF) to increase neuronal survival, differentiation, synaptic plasticity, and neurogenesis. The therapeutic potential of TrkB activation using BDNF has been demonstrated well in several preclinical models of CNS diseases, validating TrkB as a promising drug target. Therefore, we aimed to develop TrkB-specific receptor agonists by using a monoclonal antibody approach. After generation of hybridoma clones and assessment of their binding and functional activity, we identified five mouse monoclonal antibodies that show highly selective binding to TrkB and that induce robust activation of TrkB signaling. Epitope mapping studies using competition analysis showed that each of the monoclonal antibodies recognizes a unique binding site on TrkB, some of which are distinct from BDNF docking sites. These antibodies behave as true agonists based on their ability to both activate proximal and secondary signaling molecules downstream of TrkB receptors and promote neuronal survival and neurite outgrowth. The binding affinities and the functional efficacy of these antibodies are comparable to those of BDNF, whereas they do not bind to the p75 low-affinity neurotrophin receptor at all. Therefore, they could represent novel reagents to explore the pathophysiological roles of TrkB and its potential therapeutic utility in treating CNS disorders.

Induction of anti-tumor immunity by lyophilized myeloma cells secreting GM-CSF.

We have previously demonstrated that a low dose of live myeloma FO cells induced a cellular immunity against tumor without additional modulating factors. In the present study, lyophilized myeloma FO cells were used to induce anti-tumor immunity. In a myeloma vaccination model, immunization with lyophilized myeloma FO cells alone induced a slight response. However, the immunity was dramatically enhanced by myeloma FO cells transfected with a recombinant adenovirus, Adv-1/GM-CSF. The immunocytochemical staining of Adv-1/GM-CSF transfected myeloma FO cells confirmed that more than 90% of cells were positive with GM-CSF expression. Results of sandwich ELISA showed the amount of secreted GM-CSF was 240 ng/24 h per 10(6) cells. Immunization with lyophilized myeloma FO cells secreting GM-CSF prevented the tumor growth in 60% of BALB/c mice. Antibodies against myeloma FO cells were found in the sera of immunized mice. Tumor-specific T-cell response was also evaluated using cytotoxic T lymphocyte assay. In conclusion, lyophilized myeloma FO cells secreting GM-CSF can be used as a potent vaccine to induce strong and protective anti-tumor immunity.

Inhibition of the RAGE products increases survival in experimental models of severe sepsis and systemic infection.

INTRODUCTION: The receptor for advanced glycation end products (RAGE), a multi-ligand member of the immunoglobulin superfamily, contributes to acute and chronic disease processes, including sepsis. METHODS: We studied the possible therapeutic role of RAGE inhibition in the cecal ligation and puncture (CLP) model of polymicrobial sepsis and a model of systemic listeriosis using mice genetically deficient in RAGE expression or mice injected with a rat anti-murine RAGE monoclonal antibody. RESULTS: The 7-day survival rates after CLP were 80% for RAGE-/- mice (n = 15) (P < 0.01 versus wild-type), 69% for RAGE+/- mice (n = 23), and 37% for wild-type mice (n = 27). Survival benefits were evident in BALB/c mice given anti-RAGE antibody (n = 15 per group) over serum-treated control animals (P < 0.05). Moreover, delayed treatment with anti-RAGE antibody up to 24 hours after CLP resulted in a significant survival benefit compared with control mice. There was no significant increase in tissue colony counts from enteric Gram-negative or Gram-positive bacteria in animals treated with anti-RAGE antibody. RAGE-/-, RAGE+/-, and anti-RAGE antibody-treated animals were resistant to lethality from Listeria monocytogenes by almost two orders of magnitude compared with wild-type mice. CONCLUSION: Further studies are warranted to determine the clinical utility of anti-RAGE antibody as a novel treatment for sepsis.

Vaccination with viable human umbilical vein endothelial cells prevents metastatic tumors by attack on tumor vasculature with both cellular and humoral immunity.

PURPOSE: Because tumor endothelium is rarely targeted by immunity but is critically important for tumor growth, the immunity against tumor endothelium is to be developed as a novel antitumor strategy. EXPERIMENTAL DESIGN: First, viable human umbilical vein endothelial cells (HUVEC) were immunized to C57BL/6 and BALB/c mice to evoke specific CTLs as well as antibodies against tumor endothelium. Lewis lung carcinoma or myeloma cells were subsequently inoculated to evaluate the effect on tumor growth by vaccination. Second, the effect on tumor metastasis by vaccination was studied using tumor-resected mice receiving HUVEC immunization 3 days after excision. Third, the immune sera and T lymphocytes from HUVEC-immunized mice were transferred to tumor-bearing mice and added to cultured HUVECs to investigate their antiproliferative effect. RESULTS: Viable HUVEC immunization showed potent antitumor effects in Lewis lung carcinoma and myeloma tumor models. Both immune sera and CTL inhibited tumor growth and specifically suppressed proliferation of HUVECs. Particularly, tumors entirely disappeared on day 90 after tumor inoculation in four of six tumor-bearing mice receiving CTL therapy. In a metastatic tumor model, we found that the HUVEC vaccination prolonged life span from 30.9 to 41.5 days after tumor resection compared with PBS-treated mice without apparent side effects. CONCLUSIONS: Vaccination with viable HUVECs evoked both humoral and cellular immunity against tumor microvasculature, and therefore significantly inhibited tumor growth and prolonged life span of tumor-resected mice. This may provide with a novel treatment for metastatic tumors. Moreover, we have established a convenient method to evoke specific CTL against tumor angiogenesis.

Native anti-tumor responses elicited by immunization with a low dose of unmodified live tumor cells.

The present study demonstrates that immunization with a low dose of unmodified live myeloma tumor cells (FO) elicited tumor-specific immunity. BALB/c mice were vaccinated with 10(4) live dendritic cells (DC)-FO fusion cells or 10(3) live FO cells. 80% of vaccinated mice survived from the later challenge with 1 x 10(6) FO cells, whereas all control mice developed tumors. Additionally, vaccination with live FO cells gave no protection against the growth of Lewis lung carcinoma cells in C57BL/6 mice. Cellular immunity was found to be primarily responsible for anti-tumor responses. In an adoptive immune model, the development of myeloma was greatly reduced by transfusion of lymphocytes but not sera from mice immunized with FO. T cells from immunized mice also induced lysis of FO cells in the cytotoxic T lymphocyte (CTL) assay. After co-culture with FO, IFN-gamma released from immunized T helper cells increased >10-fold, while IL-4 remained unchanged in comparison with control T cells. These findings provided the first evidence that immunization with a low dose of unmodified live FO cells was safe to mice and capable of eliciting specific protective immunity against tumor growth.

Lung endothelium targeting for pulmonary embolism thrombolysis.

BACKGROUND: Pulmonary embolism occurs frequently in hospitalized patients. Thrombolytic therapy, currently used as the major treatment, has often been associated with severe bleeding complications and has thereby been life-threatening. We have developed a novel therapeutic method based on our newly created pulmonary endothelium-specific antibody. METHODS AND RESULTS: We isolated membrane proteins of rat pulmonary vascular luminal endothelium and obtained a monoclonal antibody, RE8F5, which antigen was uniquely expressed by the pulmonary capillary endothelium. In vivo biodistribution showed that RE8F5 and its urokinase conjugate were rapidly and specifically accumulated in lung. Urokinase and the conjugate were compared in rats with pulmonary, hepatic, and lower-limb embolus. In a pulmonary embolus model, the conjugate exhibited 12-fold enhanced thrombolytic potency over urokinase, whereas plasma fibrinogen and bleeding time were unaffected. In 2 other models, no significant thrombolysis was induced by the conjugate. In contrast, thrombolysis by urokinase was found to be comparable to the pulmonary embolus model. In addition, urokinase caused significant consumption of fibrinogen in all experiments. CONCLUSIONS: These data show that urokinase equipped with lung endothelium-specific antibody is an ideal treatment for pulmonary embolism, with a high efficacy of thrombolysis and low risk of bleeding.

ADAMTS-8 exhibits aggrecanase activity and is expressed in human articular cartilage.

Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family share common structural features including a disintegrin domain, a zinc metalloprotease domain, and at least one thrombospondin motif. Aberrant expression of several of these proteins has led to an understanding of their role in human disease; however, a link to function for many has not yet been made. One such uncharacterized family member, ADAMTS-8, shares significant protein sequence homology with a subgroup of ADAMTSs that includes ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-15. Each of these proteases has been shown to cleave 'aggrecanase-susceptible' site(s) within the extracellular matrix (ECM) proteoglycan aggrecan, and ADAMTS-4 and ADAMTS-5 have been postulated to play a role in the depletion of articular cartilage in osteoarthritic disease. Based on sequence relationships, in the present study we examined the ability of ADAMTS-8 to exhibit 'aggrecanase' activity. A neoepitope monoclonal antibody (MAb; AGG-C1; anti-NITEGE373) was developed and used to demonstrate the ability of ADAMTS-8 to cleave aggrecan at the aggrecanase-susceptible Glu373-Ala374 peptide bond. In addition, expression analyses demonstrated the presence of ADAMTS-8 mRNA transcripts in normal and osteoarthritic human cartilage.

Temporal associations between interleukin 22 and the extracellular domains of IL-22R and IL-10R2.

Interleukin 22 (IL-22) is a cytokine induced during both innate and adaptive immune responses. It can effect an acute phase response, implicating a role for IL-22 in mechanisms of inflammation. IL-22 requires the presence of the IL-22 receptor (IL-22R) and IL-10 receptor 2 (IL-10R2) chains, two members of the class II cytokine receptor family (CRF2), to effect signal transduction within a cell. We studied the interaction between human IL-22 and the extracellular domains (ECD) of its receptor chains in an enzyme-linked immunoabsorbant assay (ELISA)-based format, using biotinylated IL-22 (bio-IL-22) and receptor-fusions containing the ECD of a receptor fused to the Fc of hIgG1 (IL-22R-Fc and IL-10R2-Fc). IL-22 has measurable affinity for IL-22R-Fc homodimer and undetectable affinity for IL-10R2. IL-22 has substantially greater affinity for IL-22R/IL-10R2-Fc heterodimers. Further analyses involving sequential additions of receptor homodimers and cytokine indicates that the IL-10R2(ECD) binds to a surface created by the interaction between IL-22 and the IL-22R(ECD), and thereby further stabilizes the association of IL-22 within this cytokine-receptor-Fc complex. Both a neutralizing rat monoclonal antibody, specific for human IL-22, and human IL-22BP-Fc, an Fc-fusion of the secreted IL-22 binding-protein and proposed natural antagonist for IL-22, bind to similar cytokine epitopes that may overlap the binding site for IL-22R(ECD). Another rat monoclonal antibody, specific for IL-22, binds to an epitope that may overlap a separate binding site for IL-10R2(ECD). We propose, based on this data, a temporal model for the development of a functional IL-22 cytokine-receptor complex.

A monoclonal antibody against RAGE alters gene expression and is protective in experimental models of sepsis and pneumococcal pneumonia.

The RAGE (receptor for advanced glycation end products) is believed to play a role in sepsis by perpetuating inflammation. The interaction of RAGE with a variety of host-derived ligands that accumulate during stress and inflammation further induces the expression of RAGE. It was previously shown that a rat anti-RAGE monoclonal antibody protected mice from lethality in a cecal ligation and puncture model. We studied the effects of a humanized anti-RAGE monoclonal antibody in the murine pneumococcal pneumonia model of sepsis. Moreover, a gene expression analysis was performed in lung tissue of animals that underwent cecal ligation and puncture and treated with the rat anti-RAGE monoclonal antibody, compared with controls. Administration of humanized anti-RAGE mAb 6 h after intratracheal infection with Streptococcus pneumoniae improved mortality in BALB/c mice whether a 7.5 mg/kg (P < 0.01) or a 15 mg/kg dose (P < 0.01) was administered in combination with antibiotics. Gene expression analysis showed that many of the genes modulated by treatment with the anti-RAGE antibody were those that play an important role in regulating inflammation. Anti-RAGE monoclonal antibody offered a survival advantage to septic mice. This protective role in treated animals is supported by the observed gene expression profile changes of genes involved in sepsis and inflammation.

Hemodynamic effect of apelin in a canine model of acute pulmonary thromboembolism.

The peptide apelin is expressed in the pulmonary vasculature and is involved in the pathogenesis of many cardiovascular diseases. It has a biphasic role in the regulation of vasomotor tone related to the vascular endothelium. In this study, we induced acute pulmonary embolism (APE) in dogs with autologous blood clots to assess the effect of apelin on pulmonary and systemic circulation in the acute phase of APE. The expression of apelin mRNA was found to be upregulated in the lung tissue in the early several hours after APE induction and decreased at 24 h. The expression of apelin protein in the pulmonary arteries did not change within 24 h after APE, but significantly increased in the bronchial epithelial cells as early as 1h and decreased at 24 h. In normal anesthetized dogs, intravenous bolus administration of apelin significantly reduced the mean arterial pressure (MAP), but did not significantly affect the mean pulmonary arterial pressure (MPAP). In the dogs with APE, apelin decreased MPAP, whereas its impact on MAP was not significantly different from that in the control group. Taken together, the level of endogenous apelin did not change significantly in the pulmonary arterial wall, whereas its expression in the bronchial epithelium was upregulated in the early stage of APE. The effect of exogenous apelin on vasomotor tone was complicated: it resulted in differential changes in the pulmonary and systemic arterial pressures under different physiological and pathological conditions.

Affinity maturation of a humanized rat antibody for anti-RAGE therapy: comprehensive mutagenesis reveals a high level of mutational plasticity both inside and outside the complementarity-determining regions.

Antibodies that neutralize RAGE (receptor for advanced glycation end products)-ligand interactions have potential therapeutic applications in both acute and chronic diseases. We generated XT-M4, a rat anti-RAGE monoclonal antibody that has in vivo efficacy in an acute sepsis model. This antibody was subsequently humanized. To improve the affinity of this antibody for the treatment of chronic indications, we used random and targeted mutagenesis strategies in combination with ribosome and phage-display technologies, respectively, to generate libraries of XT-M4 variants. We identified a panel of single-chain Fv antibody fragments (scFv's) that was improved up to 110-fold in a homogeneous time-resolved fluorescence competition assay against parental XT-M4 immunoglobulin G (IgG). After reformatting to bivalent scFv-Fc fusions and IgGs, we observed similar gains in potency in the same assay. Further analysis of binding kinetics as IgG revealed multiple variants with subnanomolar apparent affinity that was dictated primarily by improvements in the off-rate. All variants also had improved binding to cell surface-expressed human RAGE, and all retained, or had improved, apparent affinity for mouse RAGE. F100bL in V(H) (variable region of the heavy chain) complementarity-determining region 3 (CDR3) was one of a number of key mutations that correlated with affinity improvements and was independently identified by both mutagenesis strategies. Random mutagenesis coupled with ribosome display and high-throughput screening revealed an unexpectedly high level of mutational plasticity across the whole length of the humanized scFv, suggesting greater scope for structural optimization outside of the primary antigen-combining site defined by V(H) CDR3 and V(kappa) CDR3. In summary, our comprehensive mutagenesis approach not only achieved the desired affinity maturation of XT-M4 but also defined multiple mutational hotspots across the antibody sequence, provided an insight into the specificity-determining residues of the antibody paratope, and identified additional sites within the CDR loops where human germ-line amino acids may be introduced without affecting function.

Proteomic identification of endothelial proteins isolated in situ from atherosclerotic aorta via systemic perfusion.

The functional and structural alterations of vascular endothelium contribute to the initiation, progression, and complications of atherosclerotic plaque formation, but limited information is known about the molecular composition and pathways underlying pathological changes during atherosclerosis. We have developed an affinity proteomic strategy for in situ isolation and differential mapping of vascular endothelial proteins in normal and atherosclerotic aorta tissues. The selective labeling was carried out by perfusion of the blood vessels with an active biotin reagent for covalent modification of accessible vascular endothelial proteins. The biotinylated proteins were then enriched by streptavidin affinity chromatography, separated by SDS-PAGE, and subsequently characterized by LC-MS/MS. The described procedure led to the identification of 454 distinct proteins in normal and atherosclerotic aorta tissues. A majority of the proteins are plasma membrane associated and extracellular matrix proteins, and 81 showed altered expressions in atherosclerotic aorta tissue. The differentially expressed proteins are involved in immune and inflammatory responses, cell adhesion, and lipid metabolism. The method provides a new avenue for investigating the endothelial dysfunction and development of atherosclerosis.

Efficacy of IL-13 neutralization in a sheep model of experimental asthma.

IL-13 contributes to airway hyperresponsiveness, mucus secretion, inflammation, and fibrosis, suggesting that it plays a central role in asthma pathogenesis. Neutralization of IL-13 with sIL-13Ralpha2-Fc (sIL-13R) reduces allergen-induced airway responses in rodent models of respiratory disease, but its efficacy in a large animal model has not been previously reported. In this study, we determined whether two different strategies for IL-13 neutralization modified experimental asthma in sheep. Sheep with natural airway hypersensitivity to Ascaris suum antigen were treated intravenously either with sIL-13R, a strong antagonist of sheep IL-13 bioactivity in vitro, or with IMA-638 (IgG1, kappa), a humanized antibody to human IL-13. Higher doses of IMA-638 were used because, although it is a potent antagonist of human IL-13, this antibody has 20 to 30 times lower binding and neutralization activity against sheep IL-13. Control animals received human IgG of irrelevant specificity. Sheep were treated 24 h before inhalation challenge with nebulized A. suum. The effects on antigen-induced early and late bronchial responses, and antigen-induced hyperresponsiveness, were assessed. Both sIL-13R and IMA-638 provided dose-dependent inhibition of the antigen-induced late responses and airway hyperresponsiveness. The highest dose of IMA-638 also reduced the early phase response. These findings suggest that IL-13 contributes to allergen-induced airway responses in this sheep model of asthma, and that neutralization of IL-13 is an effective strategy for blocking these A. suum-induced effects.