Martinsried, Germany
Martinsried, Germany
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Dovizio M.,University of Chieti Pescara | Maier T.J.,Frankfurt University | Alberti S.,University of Chieti Pescara | Francesco L.D.,University of Chieti Pescara | And 7 more authors.
Molecular Pharmacology | Year: 2013

Cyclooxygenase (COX)-2-derived prostanoids can influence several processes that are linked to carcinogenesis. We aimed to address the hypothesis that platelets contribute to aberrant COX-2 expression in HT29 colon carcinoma cells and to reveal the role of platelet-induced COX-2 on the expression of proteins involved in malignancy and marker genes of epithelialmesenchymal transition (EMT). Human platelets cocultured with HT29 cells rapidly adhered to cancer cells and induced COX-2 mRNA expression, but not protein synthesis, which required the late release of platelet-derived growth factor and COX-2 mRNA stabilization. Platelet-induced COX-2-dependent prostaglandin E2 (PGE2) synthesis in HT29 cells was involved in the downregulation of p21WAF1/CIP1 and the upregulation of cyclinB1 since these effects were prevented by rofecoxib (a selective COX-2 inhibitor) and rescued by exogenous PGE2. Galectin-3, which is highly expressed in HT29 cells, is unique among galectins because it contains a collagen-like domain. Thus, we studied the role of galectin-3 and platelet collagen receptors in plateletinduced COX-2 overexpression. Inhibitors of galectin-3 function (b-lactose, a dominant-negative form of galectin-3, Gal-3C, and anti-galectin-3 antibody M3/38) or collagen receptor-mediated platelet adhesion (revacept, a dimeric platelet collagen receptor GPVI-Fc) prevented aberrant COX-2 expression. Inhibition of platelet-cancer cell interaction by revacept was more effective than rofecoxib in preventing platelet-induced mRNA changes of EMT markers, suggesting that direct cell-cell contact and aberrant COX-2 expression synergistically induced gene expression modifications associated with EMT. In conclusion, our findings provide the rationale for testing blockers of collagen binding sites, such as revacept, and galectin-3 inhibitors in the prevention of colon cancer metastasis in animal models, followed by studies in patients. © 2013 by The American Society for Pharmacology.


Lippok S.,Ludwig Maximilians University of Munich | Seidel S.A.I.,Ludwig Maximilians University of Munich | Duhr S.,NanoTemper Technologies GmbH | Uhland K.,Corimmun | And 3 more authors.
Analytical Chemistry | Year: 2012

The direct quantification of both the binding affinity and absolute concentration of disease-related biomarkers in biological fluids is particularly beneficial for differential diagnosis and therapy monitoring. Here, we extend microscale thermophoresis to target immunological questions. Optically generated thermal gradients were used to deplete fluorescently marked antigens in 2- and 10-fold-diluted human serum. We devised and validated an autocompetitive strategy to independently fit the concentration and dissociation constant of autoimmune antibodies against the cardiac ?1-adrenergic receptor related to dilated cardiomyopathy. As an artificial antigen, the peptide COR1 was designed to mimic the second extracellular receptor loop. Thermophoresis resolved antibody concentrations from 2 to 200 nM and measured the dissociation constant as 75 nM. The approach quantifies antibody binding in its native serum environment within microliter volumes and without any surface attachments. The simplicity of the mix and probe protocol minimizes systematic errors, making thermophoresis a promising detection method for personalized medicine. © 2012 American Chemical Society.


Munch G.,Corimmun | Boivin-Jahns V.,University of Würzburg | Holthoff H.-P.,Corimmun | Adler K.,Corimmun | And 7 more authors.
European Journal of Heart Failure | Year: 2012

Aims A novel concept for the treatment of heart failure is the neutralization of antibodies against the β1-adrenergic receptor (anti-β1AR-ab). In a rat model of autoimmune cardiomyopathy, the cyclic peptide COR-1 (given i.v. once monthly) neutralized anti- β1AR-abs and prevented anti-β1AR-ab-induced myocardial damage, and completely reverted cardiac dysfunction over 3-6 months.Methods and resultsA clinical phase I trial was designed as a single-blinded, placebo-controlled study. Fifty human volunteers received COR-1 or matching placebo as a single i.v. administration with ascending doses (10-240 mg). Primary endpoints were safety and tolerability, while the pharmacokinetic profile of COR-1 was assessed as a secondary endpoint. All five investigated dose groups were well tolerated; no drug-related side effects occurred. Pharmacokinetics revealed a favourable profile with an almost complete plasma clearance within 60 min after administration. Pharmacodynamic investigation showed dose-dependent efficacy with almost complete scavenging of pathological anti-β1AR-abs ex vivo at the two highest doses. No anti-COR-1 autoantibodies occurred. No other effects on the immune system (such as an increase of crucial cytokines) were observed up to 43 days after drug administration, nor upon incubation of anti-β1AR-ab-positive patient blood samples with COR-1 ex vivo.ConclusionsCOR-1 was shown to be safe after i.v. administration in vivo; no relevant side effects occurred. Efficacy was estimated from ex vivo investigation of the potency to neutralize specific anti-β1-AR-abs. Trial registration: NCT 01043146, Eudra CT 2008-007745-31. © 2012 The Author.


Ungerer M.,Corimmun | Rosport K.,Corimmun | Bultmann A.,Corimmun | Piechatzek R.,ABX CRO GmbH | And 4 more authors.
Circulation | Year: 2011

Background-: Blocking of glycoprotein VI-dependent pathways by interfering in vascular collagen sites is commonly seen as an attractive target for an antiplatelet therapy of acute atherosclerotic diseases such as myocardial infarction or stroke. Revacept (soluble dimeric glycoprotein VI-Fc fusion protein) has been shown to reduce platelet adhesion by blocking vascular collagen in plaques or erosion and to be safe in preclinical studies. A dose-escalating clinical phase I study was performed to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of Revacept in humans. Methods and results-: In a first-in-humans study, 30 healthy men received a single intravenous administration of 10, 20, 40, 80, or 160 mg Revacept. The serum concentration-time courses of each dosage of Revacept showed a narrow variation and a concentration and time dependence. Revacept did not significantly affect the bleeding time. Collagen-induced platelet aggregation was dose-dependently inhibited up to 48 hours at lower doses and for 7 days after higher dose levels. In contrast, ADP- or thrombin receptor activating peptide-dependent platelet aggregation remained unaltered. There were no relevant drug-related adverse events or drug-related changes in laboratory parameters (biochemistry, hematology, and coagulation parameters). There were no drug-related changes in blood pressure, pulse rate, or ECG parameters (including 24-hour Holter monitoring). No anti-Revacept antibodies were detected. CONCLUSION-: This phase I study demonstrated that Revacept is a safe and well-tolerated new antiplatelet compound with a clear dose-dependent pharmacokinetic profile with specific, dose-related inhibition of platelet aggregation despite completely unaltered general hemostasis. Copyright © 2011 American Heart Association. All rights reserved.


Zeibig S.,Corimmun | Li Z.,Corimmun | Wagner S.,Corimmun | Holthoff H.-P.,Corimmun | And 7 more authors.
Circulation Research | Year: 2011

Rationale: There is strong evidence that oxidative modification of low-density lipoprotein (oxLDL) plays a critical role in atherogenesis and that oxLDL may profoundly influence the mechanical stability of atherosclerotic plaques. Objective: To block oxLDL, we designed, expressed, and tested Fc-CD68, a soluble oxLDL binding protein consisting of human Fc and the extracellular domain of the human oxLDL-binding receptor CD68. Methods and Results: Fc-CD68 bound with high specific affinity to oxLDL and strongly bound and colocalized with oxLDL in plaques. To study the effects of repeated administrations of Fc-CD68 on the progression of atherosclerosis and plaque vulnerability, 12- and 16-week old cholesterol-fed ApoE-/- mice received either Fc-CD68 (n=6) or Fc control protein (n=6 to 8) thrice weekly for 4 weeks. Macroscopic and histological analysis of Sudan red lipid staining showed strong and significant reduction of plaque extension in the aorta and in the aortic root, respectively. Histological analysis of pentachrome- and Sirius-stained sections of the brachiocephalic arteries of 20 week-old ApoE-/- mice revealed that Fc-CD68 significantly reduced the occurrence of spontaneous ruptures of established plaques by ≈20%, compared with Fc and drastically increased the collagen content of plaques. Furthermore, in immunostained sections of the brachiocephalic artery and the aortic root, Fc-CD68 reduced the infiltration of plaques with T lymphocytes, and macrophages by ≈50% and 30%, respectively. Conclusions: The oxLDL binding protein Fc-CD68 attenuates atherosclerosis and strengthens the stability of atherosclerotic plaques. © 2011 American Heart Association. All rights reserved.


Holthoff H.-P.,Corimmun | Zeibig S.,Corimmun | Jahns-Boivin V.,Ludwig Maximilians University of Munich | Bauer J.,Corimmun | And 7 more authors.
Circulation Research | Year: 2012

Rationale: Autoantibodies directed against the second extracellular loop of the cardiac β1-adrenergic receptor (β1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas heart disease. Various approaches have been used to detect such autoantibodies; however, the reported prevalence varies largely, depending on the detection method used. Objective: We analyzed sera from 167 DCM patients (ejection fraction <45%) and from 110 age-matched volunteers who did not report any heart disease themselves, with an often used simple peptide-ELISA approach, and compared it with a novel whole cell-based ELISA, using cells expressing the full transgene for the human β1-AR. Additionally, 35 patients with hypertensive heart disease with preserved ejection fraction were investigated. Methods and Results: The novel assay was designed according to the currently most reliable anti-TSH receptor antibody-ELISA used to diagnose Graves disease ("third-generation assay") and also detects the target antibodies by competition with a specific monoclonal anti-β1-AR antibody (β1-AR MAb) directed against the functionally relevant β1-AR epitope. Anti-β1-AR antibodies were detected in 60% of DCM patients and in 8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti-β1-AR antibody titers (defined as inhibition of β1-AR MAb-binding) were no longer detected after depleting sera from IgG antibodies by protein G adsorption. In contrast, a previously used ELISA conducted with a linear 26-meric peptide derived from the second extracellular β1-AR loop yielded a high number of false-positive results precluding any specific identification of DCM patients. Conclusions: We established a simple and efficient screening assay detecting disease-relevant β1-AR autoantibodies in patient sera yielding a high reproducibility also in high throughput screening. The assay was validated according to "good laboratory practice" and can serve as a companion biodiagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure. © 2012 American Heart Association, Inc.


Bultmann A.,Corimmun | Li Z.,Corimmun | Wagner S.,Corimmun | Peluso M.,Corimmun | And 9 more authors.
Journal of Molecular and Cellular Cardiology | Year: 2010

Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1. mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10. weeks of treatment with the anti-GPVI antibody JAQ1 (2. mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1. mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly via fibronectin-could relevantly contribute to platelet-triggered atheroprogression. © 2010 Elsevier Ltd.


Trademark
Corimmun | Date: 2011-07-12

Fusion proteins being diagnostic reagents and proteins for use in ELISA kits for scientific, research, radiology, ultrasound, sonography, plaque imaging, and nuclear medicine use; proteins for use in science, namely, protein arrays for scientific research. Pharmaceutical preparations and substances in the nature of drugs, all for the treatment and prevention of cardiovascular diseases; Veterinary preparations and substances in the nature of drugs, all for small or large animals with cardiovascular diseases; fusion proteins for pharmaceutical purposes, namely, for the treatment and prevention of cardiovascular disorders; cardiovascular treatment preparations; pharmaceutical preparations for the treatment or prevention of cardiovascular disorders.


Patent
Corimmun | Date: 2011-10-19

An isolated nucleic acid molecule selected from the group consisting of:vi. a nucleic acid molecule comprising a nucleotide sequence which is at least 85% identical to the nucleotide sequence of SEQ ID NO:1 or a complement thereof;vii. a nucleic acid molecule comprising a fragment of at least 1500 consecutive nucleotides of the nucleotide sequence of SEQ ID NO:1, or a complement thereof;viii. a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least 85% identical to SEQ ID NO:2;ix. a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 500 contiguous amino acids of SEQ ID NO: 2; andx. a nucleic acid molecule encoding a polypeptide containing a humanized immunoglobulin or parts of an immunoglubulin having binding specificity for CD133a nucleic acid molecule which encodes a variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising the entire SEQ ID NO: 1, or complement thereof under conditions of incubation at 45 C in 6.0xSSC followed by washing in 0.2xSSC/0.1 % SDS at 65 C.


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