Antibody-Mediated Neutralization of uPA Proteolytic Function Reduces Disease Progression in Mouse Arthritis Models.

Genetic absence of the urokinase-type plasminogen activator (uPA) reduces arthritis progression in the collagen-induced arthritis (CIA) mouse model to an extent just shy of disease abrogation, but this remarkable observation has not been translated into therapeutic intervention. Our aim was to test the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes mouse uPA significantly reduced arthritis progression in the CIA and delayed-type hypersensitivity arthritis models. In the CIA model, the impact of anti-uPA treatment was on par with the effect of blocking TNF-α by etanercept. A pharmacokinetics evaluation of the therapeutic Ab revealed target-mediated drug disposition consistent with a high turnover of endogenous uPA. The cellular expression patterns of uPA and uPAR were characterized by double immunofluorescence in the inflamed synovium from patients with rheumatoid arthritis and compared with synovium from healthy donors. The arthritic synovium showed expression of uPA and uPAR in neutrophils, macrophages, and a fraction of endothelial cells, whereas there was little or no expression in synovium from healthy donors. The data from animal models and human material provide preclinical proof-of-principle that validates uPA as a novel therapeutic target in rheumatic diseases.

Urokinase plasminogen activator is a central regulator of macrophage three-dimensional invasion, matrix degradation, and adhesion.

Urokinase plasminogen activator (uPA) and its receptor (uPAR) coordinate a plasmin-mediated proteolytic cascade that has been implicated in cell adhesion, cell motility, and matrix breakdown, for example, during inflammation. As part of their function during inflammatory responses, macrophages move through tissues and encounter both two-dimensional (2D) surfaces and more complex three-dimensional (3D) interstitial matrices. Based on approaches employing uPA gene-deficient macrophages, plasminogen supplementation, and neutralization with specific protease inhibitors, it is reported in this study that uPA activity is a central component of the invasion of macrophages through a 3D Matrigel barrier; it also has a nonredundant role in macrophage-mediated matrix degradation. For murine macrophages, matrix metalloproteinase-9 activity was found to be required for these uPA-mediated effects. Evidence for a unique role for uPA in the inverse relationship between macrophage adhesion and 2D migration was also noted: macrophage adhesion to vitronectin was enhanced by uPA and blocked by plasminogen activator inhibitor-1, the latter approach also able to enhance in turn the 2D migration on this matrix protein. It is therefore proposed that uPA can have a key role in the inflammatory response at several levels as a central regulator of macrophage 3D invasion, matrix remodeling, and adhesion.

Identification and preclinical development of an anti-proteolytic uPA antibody for rheumatoid arthritis.

Blocking the proteolytic capacity of urokinase-type plasminogen activator (uPA) with a monoclonal antibody (mAb) reduces arthritis progression in the collagen-induced mouse arthritis model to an extent that is on par with the effect of blocking tumor necrosis factor-alpha by etanercept. Seeking to develop a novel therapy for rheumatoid arthritis, a humanized mAb, NNC0266-0043, was selected for its dual inhibition of both the zymogen activation and the proteolytic capacity of human uPA. The antibody revealed nonlinear elimination kinetics in cynomolgus monkeys consistent with binding to and turnover of endogenous uPA. At a dose level of 20.6 mg kg-1, the antibody had a plasma half-life of 210 h. Plasma uPA activity, a pharmacodynamic marker of anti-uPA therapy, was reduced to below the detection limit during treatment, indicating that an efficacious plasma concentration was reached. Pharmacokinetic modeling predicted that sufficient antibody levels can be sustained in arthritis patients dosed subcutaneously once weekly. The anti-uPA mAb was also well tolerated in cynomolgus monkeys at weekly doses up to 200 mg kg-1 over 4 weeks. The data from cynomolgus monkeys and from human material presented here indicates that anti-uPA mAb NNC0266-0043 is suitable for clinical testing as a novel therapeutic for rheumatic diseases. KEY MESSAGES: Background: Anti-uPA therapy is on par with etanercept in a mouse arthritis model. A new humanized antibody blocks activation and proteolytic activity of human uPA. The antibody represents a radically novel mode-of-action in anti-rheumatic therapy. The antibody has PK/PD properties in primates consistent with QW clinical dosing.

The prognostic value of Stathmin-1, S100A2, and SYK proteins in ER-positive primary breast cancer patients treated with adjuvant tamoxifen monotherapy: an immunohistochemical study.

INTRODUCTION: We recently found that DNA methylation of S100A2, spleen tyrosine kinase (SYK), and Stathmin-1 (STMN1) correlates with response to tamoxifen therapy in metastatic breast cancer. In this retrospective study, we investigated immunohistochemically whether these three markers are predictors of relapse in early breast cancer (EBC) patients treated with adjuvant tamoxifen alone. METHODS: Immunohistochemical staining was performed for S100A2, SYK and STMN1 on a tissue microarray containing ER-positive invasive breast carcinomas from a study cohort of 215 operable breast cancer patients, who underwent radical local therapy and who were treated with adjuvant tamoxifen monotherapy. Cox regression was used to correlate staining intensity of the three markers with main endpoints in our study; disease-free survival (DFS), and disease-specific survival (DSS). RESULTS: In univariate analysis, only STMN1 staining intensity strongly correlated with DFS (P = 0.014) and DSS (P = 0.002). In the groups of low and high STMN1 intensity, DFS was 84% and 63%, and DSS was 89% and 70%. STMN1 retained its prognostic value for DFS (P = 0.002) and DSS (<0.001) in the multivariate model together with lymph node status. We found also a trend to better DFS in patients with low STMN1 intensity in both lymph node-positive (P = 0.001) and -negative patients (P = 0.065). As the tumour cells did not express S100A2 (except in one case) the potential prognostic value of this marker was not evaluated. CONCLUSIONS: Staining intensity of STMN1, but not SYK, predicted outcome in our collective of ER- positive tamoxifen treated EBC patients.

Urokinase plasminogen activator and receptor promote collagen-induced arthritis through expression in hematopoietic cells.

The plasminogen activation (PA) system has been implicated in driving inflammatory arthritis, but the precise contribution of PA system components to arthritis pathogenesis remains poorly defined. Here, the role of urokinase plasminogen activator (uPA) and its cognate receptor (uPAR) in the development and severity of inflammatory joint disease was determined using uPA- and uPAR-deficient mice inbred to the strain DBA/1J, a genetic background highly susceptible to collagen-induced arthritis (CIA). Mice deficient in uPA displayed a near-complete amelioration of macroscopic and histological inflammatory joint disease following CIA challenge. Similarly, CIA-challenged uPAR-deficient mice exhibited significant amelioration of arthritis incidence and severity. Reduced disease development in uPA-deficient and uPAR-deficient mice was not due to an altered adaptive immune response to the CIA challenge. Reciprocal bone marrow transplant studies indicated that uPAR-driven CIA was due to expression by hematopoietic-derived cells, as mice with uPAR-deficient bone marrow challenged with CIA developed significantly reduced macroscopic and histological joint disease as compared with mice with uPAR expression limited to non-hematopoietic-derived cells. These findings indicate a fundamental role for uPAR-expressing hematopoietic cells in driving arthritis incidence and progression. Thus, uPA/uPAR-mediated cell surface proteolysis and/or uPAR-mediated signaling events promote inflammatory joint disease, indicating that disruption of this key proteolytic/signaling system may provide a novel therapeutic strategy to limit clinical arthritis.

Characterization of anti-TIMP-1 monoclonal antibodies for immunohistochemical localization in formalin-fixed, paraffin-embedded tissue.

The aim of this study was to evaluate seven anti-TIMP-1 (tissue inhibitor of metalloproteinase-1) monoclonal antibodies by immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissue. Detection of the TIMP-1 protein was studied by IHC in FFPE human archival normal and neoplastic samples. Indirect IHC technique was used, and the seven antibodies (clones VT1, VT2, VT4, VT5, VT6, VT7, and VT8) were tested in various concentrations using different pretreatment protocols. All seven VT antibodies specifically immunostained the cytoplasm of islets of Langerhans cells in normal pancreas, epithelial cells of hyperplastic prostate, tumor cells of medullary thyroid carcinoma, and fibroblast-like cells of malignant melanoma. Specificity of the anti-TIMP-1 antibodies was confirmed by several controls, e.g., Western blotting on proteins extracted from FFPE tissue showed that the VT7 antibody reacted specifically with a protein band of approximately 28 kDa, corresponding to the molecular mass of TIMP-1. However, sensitivity varied with the different antibodies. Use of heat-induced epitope retrieval (HIER) and the VT7 clone applied at low concentrations demonstrated more intense immunoreactivity with the TIMP-1-positive cell types compared to the other six clones. Furthermore, when tested on a range of normal and neoplastic endocrine tissues, the VT7 clone demonstrated immunoreactivity with all neuroendocrine cell types. In conclusion, all seven antibodies detected TIMP-1 protein in various normal and neoplastic FFPE tissues, but one clone, VT7, was superior for IHC staining of TIMP-1 in FFPE tissue sections when using HIER.

Treatment with anti-C5aR mAb leads to early-onset clinical and mechanistic effects in the murine delayed-type hypersensitivity arthritis model.

Blockade of the complement cascade at the C5a/C5a receptor (C5aR)-axis is believed to be an attractive treatment avenue in rheumatoid arthritis (RA). However, the effects of such interventions during the early phases of arthritis remain to be clarified. In this study we use the murine delayed-type hypersensitivity arthritis (DTHA) model to study the very early effects of a blocking, non-depleting anti-C5aR mAb on joint inflammation with treatment synchronised with disease onset, an approach not previously described. The DTHA model is a single-paw inflammatory arthritis model characterised by synchronised and rapid disease onset driven by T-cells, immune complexes and neutrophils. We show that a reduction in paw swelling, bone erosion, cartilage destruction, synovitis and new bone formation is apparent as little as 60 h after administration of a single dose of a blocking, non-depleting anti-mouse C5aR mAb. Importantly, infiltration of neutrophils into the joint and synovium is also reduced following a single dose, demonstrating that C5aR signalling during the early stage of arthritis regulates neutrophil infiltration and activation. Furthermore, the number of T-cells in circulation and in the draining popliteal lymph node is also reduced following a single dose of anti-C5aR, suggesting that modulation of the C5a/C5aR axis results in effects on the T cell compartment in inflammatory arthritis. In summary, these data demonstrate that blockade of C5aR leads to rapid and significant effects on arthritic disease development in a DTHA model strengthening the rationale of C5aR-blockade as a treatment strategy for RA, especially during the early stages of arthritis flare.

Sensitive and specific in situ hybridization for early drug discovery.

High-throughput analyses of gene expression such as microarrays and RNA-sequencing are widely used in early drug discovery to identify disease-associated genes. To further characterize the expression of selected genes, in situ hybridization (ISH) using RNA probes (riboprobes) is a powerful tool to localize mRNA expression at the cellular level in normal and diseased tissues, especially for novel drug targets, where research tools like specific antibodies are often lacking.We describe a sensitive ISH protocol using radiolabelled riboprobes suitable for both paraffin-embedded and cryo-preserved tissue. The riboprobes are generated by in vitro transcription using PCR products as templates, which is less time consuming compared to traditional transcription from linearized plasmids, and offers a relatively simple way to generate several probes per gene, e.g., for splice variant analyses. To ensure reliable ISH results, we have incorporated a number of specificity controls in our standard experimental setup. We design antisense probes to cover two non-overlapping parts of the gene of interest, and use the corresponding sense probes as controls for unspecific binding. Probes are furthermore tested on sections of paraffin-embedded or cryo-preserved positive and negative control cells with known gene expression. Our protocol thus provides a method for sensitive and specific ISH, which is suitable for target validation and characterization in early drug discovery.

Establishment and characterization of a sustained delayed-type hypersensitivity model with arthritic manifestations in C57BL/6J mice.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic progressive, inflammatory and destructive autoimmune disease, characterised by synovial joint inflammation and bone erosion. To better understand the pathophysiology and underlying immune mechanisms of RA various models of arthritis have been developed in different inbred strains of mice. Establishment of arthritis models with components of adaptive immunity in the C57BL/6J strain of mice has been difficult, and since most genetically modified mice are commonly bred on this background, there is a need to explore new ways of obtaining robust models of arthritis in this strain. This study was undertaken to establish and characterise a novel murine model of arthritis, the delayed-type hypersensitivity (DTH)-arthritis model, and evaluate whether disease can be treated with compounds currently used in the treatment of RA. METHODS: DTH-arthritis was induced by eliciting a classical DTH reaction in one paw with methylated bovine serum albumin (mBSA), with the modification that a cocktail of type II collagen monoclonal antibodies was administered between the immunisation and challenge steps. Involved cell subsets and inflammatory mediators were analysed, and tissue sections evaluated histopathologically. Disease was treated prophylactically and therapeutically with compounds used in the treatment of RA. RESULTS: We demonstrate that DTH-arthritis could be induced in C57BL/6 mice with paw swelling lasting for at least 28 days and that disease induction was dependent on CD4+ cells. We show that macrophages and neutrophils were heavily involved in the observed pathology and that a clear profile of inflammatory mediators associated with these cell subsets was induced locally. In addition, inflammatory markers were observed systemically. Furthermore, we demonstrate that disease could be both prevented and treated. CONCLUSIONS: Our findings indicate that DTH-arthritis shares features with both collagen-induced arthritis (CIA) and human RA. DTH-arthritis is dependent on CD4+ cells for induction and can be successfully treated with TNFα-blocking biologics and dexamethasone. On the basis of our findings we believe that the DTH-arthritis model could hold potential in the preclinical screening of novel drugs targeting RA. The model is highly reproducible and has a high incidence rate with synchronised onset and progression, which strengthens its potential.

Concentrations of TIMP1 mRNA splice variants and TIMP-1 protein are differentially associated with prognosis in primary breast cancer.

BACKGROUND: TIMP-1 protein is a prognostic factor for recurrence-free and overall survival (OS) time in breast cancer. We evaluated the prognostic value of TIMP1 mRNA and a novel TIMP1 mRNA splice variant in 1301 primary breast cancer patients. METHODS: We measured mRNA transcripts of full-length TIMP1 (TIMP1-v1) and the novel splice variant lacking exon 2 (TIMP1-v2) by use of real-time RT-PCR in frozen primary tumor samples. Transcript concentrations are correlated with histomorphological and biological factors, TIMP-1 protein, and distant metastasis-free survival (MFS) and OS time. RESULTS: TIMP1-v1 and TIMP1-v2 alone were not informative with respect to predicting prognosis. However, the PCR assay designed to measure the combination of v1 + v2 showed that high concentrations of this combination were associated with good prognosis. In Cox multivariate regression analysis, which also included the traditional prognostic factors, increasing concentrations were independently associated with prolonged MFS (P = 0.004) and OS (P = 0.048). Including TIMP-1 protein and TIMP1-v1+v2 mRNA together in the multivariate model revealed that protein and mRNA were both independently associated with prognosis, with hazard ratios pointing in opposite directions. CONCLUSION: High concentrations of TIMP1-v1+2 mRNA are associated with good prognosis in patients with primary breast cancer. Since high concentrations of TIMP-1 protein are associated with poor prognosis, the presence of possible posttranscriptional mechanisms requires further investigation.

Expression of urokinase plasminogen activator, its receptor and type-1 inhibitor in malignant and benign prostate tissue.

The plasminogen activation (PA) cascade participates in degradation of extracellular matrix during cancer invasion. We have studied the expression of urokinase-type plasminogen activator (uPA) mRNA, uPA receptor (uPAR) mRNA and immunoreactivity, and type-1 plasminogen activator inhibitor (PAI-1) mRNA and immunoreactivity in 16 prostate adenocarcinomas and 9 benign prostate hyperplasias. uPA mRNA and uPAR mRNA expression were found in 9 and 8 of the adenocarcinomas, respectively, and in 7 and 6 of the benign hyperplasias, respectively. In both malignant and benign lesions, expression of these 2 mRNAs was predominantly seen in cells identified as macrophages, which in most of the carcinomas (approximately 90%) were located in the interstitial tissue between the tumor cell islands, while in most of the benign hyperplasias they were located in the lumen of the glands and were in only a few cases (approximately 30%) found in the interstitial tissue. uPAR immunoreactivity correlated with the mRNA expression and was, in addition, found in neutrophils. PAI-1 mRNA was detected in 13 of the 16 carcinomas and in 8 of the 9 benign hyperplasias, located in scattered fibroblast-like cells in both groups, in some vascular structures and in a few macrophages located in the interstitial tissue of both malignant and benign lesions. A similar expression pattern was found for PAI-1 immunoreactivity. In 8 of the 16 carcinomas, all 3 components were present, and in several areas colocalization was observed in stromal cells in close proximity to cancer cell islands. No immunoreactivity and/or mRNA expression of uPA, uPAR or PAI-1 was observed in cancer cells or in other epithelial cells in any of the cases.