CTLA4-Ig prevents development of neutralizing antibody formation after continuous treatment with human FVIII in HA rats.

INTRODUCTION: Immunogenicity causing development of anti-drug antibodies (ADAs) are major challenges in the treatment of haemophilia, as well as other diseases where proteins are used for treatment. Furthermore, it is a complication for preclinical testing of such therapies in animal models. AIM: To investigate if the immunosuppressive drug CTLA4 immunoglobulin (CTLA4-Ig) can induce tolerance in haemophilia A (HA) rats receiving recombinant human coagulation factor VIII (rhFVIII) treatment. METHODS: Two different prophylactic rhFVIII compounds were given intravenously to HA rats for 4 weeks. Both rhFVIII compounds were co-administered with commercially available CTLA4-Ig or human IgG subclass 4 (hIgG4) as control, and blood samples were collected. To functionally test if pharmacological efficacy was retained, rats were subjected to a bleeding experiment under anaesthesia at end of study. RESULTS: The mean inhibitory level after 4 weeks in rats receiving rhFVIII and hIgG4 was 85.7 BU for octocog alfa and 37.4 BU for rurioctocog alfa pegol. In contrast, co-administration with CTLA4-Ig during rhFVIII therapy prevented the formation of ADAs (both binding and inhibitory) in 14/14 rats receiving octocog alfa and in 7/7 rats receiving rurioctocog alfa pegol. Moreover, we were able to show that the pharmacological efficacy of rhFVIII was preserved. CONCLUSION: In a rat model with spontaneous bleeding, co-administration of CTLA4-Ig with rhFVIII prevented antibody formation. No FVIII antibodies were detected, demonstrating that CTLA4-Ig co-administration can be applicable as a method to prevent immunogenicity, when evaluating human proteins in preclinical systems permitting continuous pharmacokinetic and pharmacodynamic assessment.

Normal Neurodevelopment and Fertility in Juvenile Male Rats Exposed to Polyethylene Glycol Following Dosing With PEGylated rFIX (Nonacog Beta Pegol, N9-GP): Evidence from a 10-Week Repeat-Dose Toxicity Study.

N9-GP/Rebinyn®/Refixia® is an approved PEGylated (polyethylene glycol-conjugated) recombinant human factor IX intended for prophylactic and/or on-demand treatment in adults and children with haemophilia B. A juvenile neurotoxicity study was conducted in male rats to evaluate effects on neurodevelopment, sexual maturation, and fertility following repeat-dosing of N9-GP. Male rats were dosed twice weekly from Day 21 of age with N9-GP or vehicle for 10 weeks, followed by a dosing-free recovery period for 13 weeks and terminated throughout the dosing and recovery periods. Overall, dosing N9-GP to juvenile rats did not result in any functional or pathological effects, as measured by neurobehavioural/neurocognitive tests, including motor activity, sensory function, learning and memory as well as growth, sexual maturation, and fertility. This was further supported by the extensive histopathologic evaluation of brain tissue. Exposure and distribution of polyethylene glycol was investigated in plasma, choroid plexus, cerebrospinal fluid, and brain sections. PEG did not cross the blood brain barrier and PEG exposure did not result in any effects on neurodevelopment. In conclusion, dosing of N9-GP to juvenile rats did not identify any effects on growth, sexual maturation and fertility, clinical and histological pathology, or neurodevelopment related to PEG exposure and supports the prophylactic use of N9-GP in children.

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 receptor cleavage correlates with tumor volume in a transgenic mouse model of breast cancer.

The urokinase plasminogen activator system plays a key role in tissue degradation during cancer invasion. The linker region between domains I and II of the intact, three domain urokinase receptor uPAR(I-III) is highly susceptible to proteolytic cleavage and the resulting cleaved uPAR forms are strong prognostic biomarkers in several types of cancer, i.e., high levels of the cleaved uPAR forms indicate poor survival. To better understand the role of uPAR cleavage in cancer, we have designed immunoassays for specific quantification of intact mouse uPAR [muPAR(I-III)] and mouse uPAR domain I [muPAR(I)]. The level of muPAR(I) is significantly increased in mammary tumor-bearing mice compared to controls and, notably, there is a strong correlation to tumor volume. In contrast, the tumor volume is only weakly correlated to the level of intact muPAR(I-III), indicating that cleavage of muPAR is a more specific marker for cancer than increased expression of muPAR per se. The levels of the muPAR forms are dramatically affected by in vivo challenge with a urokinase -blocking antibody, demonstrating a functional role of uPA in uPAR cleavage. The levels of the muPAR forms are, however, unaffected by uPA-deficiency, suggesting that redundant proteases maintains the task of cleaving uPAR(I-III) when uPA is absent. Our findings emphasize the significance of the cleaved uPAR forms as cancer biomarkers. The strong correlation between muPAR(I) and the tumor volume in our experimental setup may motivate investigations of human uPAR(I) as biomarker for response to oncological treatment.

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.

Evaluation of Gremlin-1 as a therapeutic target in metabolic dysfunction-associated steatohepatitis.

Gremlin-1 has been implicated in liver fibrosis in metabolic dysfunction-associated steatohepatitis (MASH) via inhibition of bone morphogenetic protein (BMP) signalling and has thereby been identified as a potential therapeutic target. Using rat in vivo and human in vitro and ex vivo model systems of MASH fibrosis, we show that neutralisation of Gremlin-1 activity with monoclonal therapeutic antibodies does not reduce liver inflammation or liver fibrosis. Still, Gremlin-1 was upregulated in human and rat MASH fibrosis, but expression was restricted to a small subpopulation of COL3A1/THY1+ myofibroblasts. Lentiviral overexpression of Gremlin-1 in LX-2 cells and primary hepatic stellate cells led to changes in BMP-related gene expression, which did not translate to increased fibrogenesis. Furthermore, we show that Gremlin-1 binds to heparin with high affinity, which prevents Gremlin-1 from entering systemic circulation, prohibiting Gremlin-1-mediated organ crosstalk. Overall, our findings suggest a redundant role for Gremlin-1 in the pathogenesis of liver fibrosis, which is unamenable to therapeutic targeting.

Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling.

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9, gelatinase B) have separately been recognized to play important roles in various tissue remodeling processes. In this study, we demonstrate that deficiency for MMP9 in combination with ablation of either uPA- or tissue-type plasminogen activator (tPA)-catalyzed plasminogen activation is critical to accomplish normal gestation in mice. Gestation was also affected by simultaneous lack of MMP9 and the uPA receptor (uPAR). Interestingly, uPA-deficiency additionally exacerbated the effect of MMP9-deficiency on bone growth and an additive effect caused by combined lack in MMP9 and uPA was observed during healing of cutaneous wounds. By comparison, MMP9-deficiency combined with absence of either tPA or uPAR resulted in no significant effect on wound healing, indicating that the role of uPA during wound healing is independent of uPAR, when MMP9 is absent. Notably, compensatory upregulation of uPA activity was seen in wounds from MMP9-deficient mice. Taken together, these studies reveal essential functional dependency between MMP9 and uPA during gestation and tissue repair.

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.

Spontaneous metastasis in matrix metalloproteinase 3-deficient mice.

Matrix metalloproteinases (MMPs) have been linked to the metastatic potential of tumor cells due to their ability to degrade the extracellular matrix. MMP-3 (stromelysin-1) is upregulated in a wide variety of human tumors. We used the MMTV-PyMT breast cancer model to determine if MMP-3 is involved in tumorigenesis and metastatic growth. In this model the stromal expression of MMP-3 mRNA resembles the predominant MMP-3 expression pattern observed in human ductal breast carcinomas. We studied a cohort of 63 PyMT transgenic mice, either deficient for MMP-3 or wild-type controls. The degree of metastasis did not differ significantly between the two groups of mice, although the median lung metastasis volume was more than threefold increased in MMTV-PyMT mice deficient in MMP-3. Likewise, primary tumor growth rate and lymph node metastasis were not significantly affected by MMP-3-deficiency. By comparing mRNA levels in MMP-3-deficient PyMT tumors with PyMT wild-type tumors we excluded compensatory transcriptional changes of other MMPs or their specific inhibitors. Thus, we conclude that genetic ablation of MMP-3 does not significantly affect tumor growth and metastasis in the MMTV-PyMT model.

Metastasis is strongly reduced by the matrix metalloproteinase inhibitor Galardin in the MMTV-PymT transgenic breast cancer model.

Matrix metalloproteinases (MMP) have several roles that influence cancer progression and dissemination. However, low molecular weight metalloproteinase inhibitors (MPI) have not yet been tested in transgenic/spontaneous metastasis models. We have tested Galardin/GM6001, a potent MPI that reacts with most MMPs, in the MMTV-PymT transgenic breast cancer model. We followed a cohort of 81 MMTV-PymT transgenic mice that received Galardin, placebo, or no treatment. Galardin treatment was started at age 6 weeks with 100 mg/kg/d, and all mice were killed at age 13.5 weeks. Galardin treatment significantly reduced primary tumor growth. Final tumor burden in Galardin-treated mice was 1.69 cm3 compared with 3.29 cm3 in placebo-treated mice (t test, P = 0.0014). We quantified the total lung metastasis volume in the same cohort of mice. The median metastasis volume was 0.003 mm(3) in Galardin-treated mice compared with 0.56 mm(3) in placebo-treated mice (t test, P < 0.0001). Thus, metastasis burden was reduced more than 100-fold, whereas primary tumor size was reduced only 2-fold. We also found that primary tumors from Galardin-treated mice exhibited a lower histopathologic tumor grade, increased collagen deposition, and increased MMP-2 activity. MMPs are known to have tumor-promoting and tumor-inhibitory effects, and several clinical trials of broad-spectrum MPIs have failed to show promising effects. The very potent antimetastatic effect of Galardin in the MMTV-PymT model does, however, show that it may be possible to find broad-spectrum MPIs with favorable inhibition profiles, or perhaps combinations of monospecific MPIs, for future clinical application.

Tumor cell-derived and macrophage-derived cathepsin B promotes progression and lung metastasis of mammary cancer.

Proteolysis in close vicinity of tumor cells is a hallmark of cancer invasion and metastasis. We show here that mouse mammary tumor virus-polyoma middle T antigen (PyMT) transgenic mice deficient for the cysteine protease cathepsin B (CTSB) exhibited a significantly delayed onset and reduced growth rate of mammary cancers compared with wild-type PyMT mice. Lung metastasis volumes were significantly reduced in PyMT;ctsb(+/-), an effect that was not further enhanced in PyMT;ctsb(-/-) mice. Furthermore, lung colonization studies of PyMT cells with different CTSB genotypes injected into congenic wild-type mice and in vitro Matrigel invasion assays confirmed a specific role for tumor-derived CTSB in invasion and metastasis. Interestingly, cell surface labeling of cysteine cathepsins by the active site probe DCG-04 detected up-regulation of cathepsin X on PyMT;ctsb(-/-) cells. Treatment of cells with a neutralizing anti-cathepsin X antibody significantly reduced Matrigel invasion of PyMT;ctsb(-/-) cells but did not affect invasion of PyMT;ctsb(+/+) or PyMT;ctsb(+/-) cells, indicating a compensatory function of cathepsin X in CTSB-deficient tumor cells. Finally, an adoptive transfer model, in which ctsb(+/+), ctsb(+/-), and ctsb(-/-) recipient mice were challenged with PyMT;ctsb(+/+) cells, was used to address the role of stroma-derived CTSB in lung metastasis formation. Notably, ctsb(-/-) mice showed reduced number and volume of lung colonies, and infiltrating macrophages showed a strongly up-regulated expression of CTSB within metastatic cell populations. These results indicate that both cancer cell-derived and stroma cell-derived (i.e., macrophages) CTSB plays an important role in tumor progression and metastasis.

Absence of functional compensation between coagulation factor VIII and plasminogen in double-knockout mice.

Plasminogen deficiency is associated with severely compromised fibrinolysis and extravascular deposition of fibrin. In contrast, coagulation factor VIII (FVIII) deficiency leads to prolonged and excessive bleeding. Based on opposing biological functions of plasminogen and FVIII deficiencies, we hypothesized that genetic elimination of FVIII would alleviate the systemic formation of fibrin deposits associated with plasminogen deficiency and, in turn, elimination of plasminogen would limit bleeding symptoms associated with FVIII deficiency. Mice with single and combined deficiencies of FVIII (F8-/-) and plasminogen (Plg-/-) were evaluated for phenotypic characteristics of plasminogen deficiency, including wasting disease, shortened lifespan, rectal prolapse, and multiorgan fibrin deposition. Conversely, to specifically examine the role of plasmin-mediated fibrinolysis on bleeding caused by FVIII deficiency, F8-/- and F8-/-/Plg-/- mice were subjected to a bleeding challenge. Mice with a combined deficiency in FVIII and plasminogen displayed no phenotypic differences relative to mice with single FVIII or plasminogen deficiency. Plg-/- and F8-/-/Plg-/- mice exhibited the same penetrance and severity of wasting disease, rectal prolapse, extravascular fibrin deposits, and reduced viability. Furthermore, following a tail vein-bleeding challenge, no significant differences in bleeding times or total blood loss could be detected between F8-/- and F8-/-/Plg-/- mice. Moreover, F8-/- and F8-/-/Plg-/- mice responded similarly to recombinant FVIII (rFVIII) therapy. In summary, the pathological phenotype of Plg-/- mice developed independently of FVIII-dependent coagulation, and elimination of plasmin-driven fibrinolysis did not play a significant role in a nonmucosal bleeding model in hemophilia A mice.

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.

Metastasis of transgenic breast cancer in plasminogen activator inhibitor-1 gene-deficient mice.

The plasminogen activator inhibitor-1 (PAI-1) blocks the activation of plasmin(ogen), an extracellular protease vital to cancer invasion. PAI-1 is like the corresponding plasminogen activator uPA (urokinase-type plasminogen activator) consistently expressed in human breast cancer. Paradoxically, high levels of PAI-1 as well as uPA are equally associated with poor prognosis in cancer patients. PAI-1 is thought to play a vital role for the controlled extracellular proteolysis during tumor neovascularization. We have studied the effect of PAI-1 deficiency in a transgenic mouse model of metastasizing breast cancer. In these tumors, the expression pattern of uPA and PAI-1 resembles that of human ductal breast cancer and plasminogen is required for efficient metastasis. In a cohort of 63 transgenic mice that were either PAI-1-deficient or wild-type sibling controls, primary tumor growth and vascular density were unaffected by PAI-1 status. PAI-1 deficiency also did not significantly affect the lung metastatic burden. These results agree with the virtual lack of spontaneous phenotype in PAI-1-deficient mice and humans and may reflect that the plasminogen activation reaction is not rate limiting for tumor vascularization and metastasis, or that there is a functional redundancy between PAI-1 and other inhibitors of the uPA/plasmin system, masking the effect of PAI-1 deficiency.

Indication of a role of plasminogen activator inhibitor type I in protecting murine fibrosarcoma cells against apoptosis.

In a number of cancer types high tumor tissue levels of plasminogen activator inhibitor type 1 (PAI-1) protein are strongly associated with shorter cancer patient survival. This association has been intriguing since PAI-1 is known to inhibit urokinase plasminogen activator (uPA) that converts plasminogen to plasmin, which is actively involved in tumor progression and invasion. In order to further explore the biological role of PAI-1 in cancer, we have prepared fibroblasts from PAI-1 gene deficient mice and from their wild type littermates. From these fibroblasts fibrosarcoma cell lines were established and characterized. Both types of fibroblasts underwent spontaneous transformation as indicated by aneuploidy, immortalization, clonogenicity in soft agar and tumor formation in vivo. While both PAI-1 deficient and PAI-1 expressing cell lines showed similar proliferation rates in vitro, cells devoid of PAI-1 were significantly more sensitive to apoptotic stimuli. When inoculated subcutaneously into nude mice PAI-1 expressing cells rapidly established tumors, while PAI-1 deficient cells had a significantly longer lag-phase before they started to grow (p<0.0001). The present study suggests that PAI-1, besides its uPA inhibiting function, has a role in cancer progression by protecting tumor cells from undergoing apoptosis.

Changes in intracellular cAMP reported by a Redistribution assay using a cAMP-dependent protein kinase-green fluorescent protein chimera.

We report on a novel method to monitor changes in intracellular cAMP concentration ([cAMP]i) within intact living cells using a chimeric fusion of the catalytic subunit of cAMP-dependent protein kinase to green fluorescent protein (PKAcat-GFP). In stably transfected unstimulated fibroblasts, fusion protein fluorescence is highly concentrated in aggregates throughout the cytoplasm and absent in the nucleus. Elevation of [cAMP]i disperses GFP fluorescence from the cytoplasmic aggregates within minutes. Spot-photobleach measurements show that the rate of exchange of GFP-labeled catalytic subunits at these aggregates increases in proportion to [cAMP]i. For any given stimulus, the response curve for dispersal of GFP fluorescence from aggregates agrees closely with the increase in total [cAMP]i as measured by standard in vitro methods (SPA). The redistribution of fluorescence is completely reversible: reduction of [cAMP]i results in return of fluorescence to the cytoplasmic aggregates. Consistent behaviour of PKAcat-GFP is seen in different cell backgrounds. We demonstrate that PKA Redistribution assays are suitable for measurement of changes in [cAMP]i brought about by both Gs- and Gi-protein-coupled receptor stimulation as well as by inhibition of cAMP phosphodiesterases.

Spontaneous lung and lymph node metastasis in transgenic breast cancer is independent of the urokinase receptor uPAR.

Urokinase-type plasminogen activator (uPA) is an extracellular protease that plays a pivotal role in tumor progression. uPA activity is spatially restricted by its anchorage to high-affinity uPA receptors (uPAR) at the cell surface. High tumor tissue expression of uPA and uPAR is associated with poor prognosis in lung, breast, and colon cancer patients in clinical studies. Genetic deficiency of uPA leads to a significant reduction in metastases in the murine transgenic MMTV-PyMT breast cancer model, demonstrating a causal role for uPA in cancer dissemination. To investigate the role of uPAR in cancer progression, we analyze the effect of uPAR deficiency in the same cancer model. uPAR is predominantly expressed in stromal cells in the mouse primary tumors, similar to human breast cancer. In a cohort of MMTV-PyMT mice [uPAR-deficient (n = 31) or wild type controls (n = 33)], tumorigenesis, tumor growth, and tumor histopathology were not significantly affected by uPAR deficiency. Lung and lymph node metastases were also not significantly affected by uPAR deficiency, in contrast to the significant reduction seen in uPA-deficient mice. Taken together, our data show that the genetic absence of uPAR does not influence the outcome of the MMTV-PyMT cancer model.

Stromal cell involvement in cancer.

Solid tumors co-opt the body's endogenous extracellular proteolytic machinery for their invasion and metastasis. This is supported by a large number of independent observations ranging from histochemical and prognostic studies of cancer patient material to animal experiments. There are several extracellular proteolytic systems that are relevant in the context of cancer, but the plasminogen activation (PA) system and the matrix metalloproteases (MMPs) remain the most thoroughly investigated. Localization studies by immunohistochemistry and in situ mRNA hybridization in tumors of common human cancers have repeatedly identified members of the PA and MMP systems in stromal cells. The cancer cells, of epithelial origin, contribute PA and MMP components in some cases, but their contribution fades in comparison with the overwhelming expression of proteolytic components by fibroblasts, macrophages, endothelial cells, and other stromal cells. Ideal animal models of human cancers should recapitulate this fundamental proteolytic aspect of tumor biology. However, in the transplantable tumor models where PA or MMP components have been studied at the cellular level in vivo, this is most often not the case. Transgenic cancer models may provide a closer parallel to the human situation, in that PA and MMP components are synthesized by the tumor stroma. The pivotal role of stromal cells has been confirmed experimentally in mouse models in which the expression pattern of proteolytic components is strongly reminiscent of human tumors. In these models it is possible to reconstitute the wild-type tumor characteristics of proteolytically deficient tumor-bearing mice by transplantation with wild-type fibroblasts or hemapoietic cells. These studies collectively show that cancer-associated proteolysis is a collaborative effort of malignant cancer cells and various stromal cells--a collaboration in which stromal cells contribute the majority of the active proteolytic components that are necessary for the invasive behavior of the tumors. This cellular division of labor positions the stromal cells as prime targets for future research and possibly therapy. Vascular endothelial cells are already the focus of intense therapeutically relevant research, but tumor-associated fibroblasts, macrophages, neutrophils, lymphendothelial cells, etc. provide additional largely unexplored territory in the ongoing search for efficient countermeasures against invasive cancer.

Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz-type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor.

INTRODUCTION: A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab. MATERIALS AND METHODS: Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats. RESULTS AND CONCLUSIONS: Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism.

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.