Karlheinz Peter

Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia

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Publications (128)689.32 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microparticles or small-molecule drugs is attracting major interest for molecular imaging and drug delivery. Nondirected bioconjugation techniques may impair antibody affinity, result in lower amounts of functional antibodies and generate multicomponent mixtures. We present a detailed protocol for the enzymatic bioconjugation of small recombinant antibodies to imaging particles, and we also describe the generation of and conjugation to a low-fouling capsule assembled for drug delivery from PEG and PVPON (poly(N-vinylpyrrolidone) by a layer-by-layer (LbL) technique. The single-chain variable fragment (scFv) is equipped with a short C-terminal LPETG tag and the fusion partners are functionalized with an N-terminal GGG nucleophilic group for sortase A conjugation. The LbL capsules are assembled through hydrogen bonding by depositing alkyne-modified poly(vinylpyrrolidone) and poly(methacrylic acid) layers on silica particles, followed by depositing alkyne-modified PEG. The generation of the antibodies and LbL capsules takes ∼1-2 weeks each. The conjugation and functional testing takes another 3-4 d.
    Nature Protocols 01/2015; 10(1):90-105. · 7.96 Impact Factor
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    ABSTRACT: Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site-specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one-pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single-chain antibody (scFv), which binds to the ligand-induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events.
    Angewandte Chemie International Edition 12/2014; · 11.34 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site-specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one-pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single-chain antibody (scFv), which binds to the ligand-induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events.
    Angewandte Chemie 12/2014;
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    ABSTRACT: Imaging of activated platelets using an activation specific anti GPIIb/IIIa integrin single-chain antibody (scFvanti-LIBS) conjugated to a positron emitting copper-64 complex of a cage amine sarcophagine chelator (MeCOSar) is reported. This tracer was compared in vitro to a 64CuII complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabelled with 64CuII rapidly under mild conditions and with higher specific activity than scFvanti-LIBS-DOTA. The utility of scFvanti-LIBS-MeCOSar as a diagnostic agent was assessed in vivo in a mouse model of acute thrombosis. The uptake of scFvanti-LIBS-64CuMeCOSar in the injured vessel was significantly higher than the non-injured vessel. Positron-emission tomography was used to show accumulation of scFvanti-LIBS-64CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-64CuMeCOSar is an excellent tool for highly sensitive in vivo detection of activated platelets in PET and has the potential to be used for early diagnosis of acute thrombotic events.
    Molecular Pharmaceutics 07/2014; · 4.57 Impact Factor
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    ABSTRACT: The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo.
    Circulation 07/2014; 130(1):35-50. · 15.20 Impact Factor
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    ABSTRACT: -Inflammation and myocardial necrosis play important roles in ischemia/reperfusion injury after coronary artery occlusion and recanalization. The detection of inflammatory activity and the extent of myocardial necrosis itself are of great clinical and prognostic interest. We developed a dual, non-invasive imaging approach using molecular magnetic resonance imaging (MRI) in an in vivo mouse model of myocardial ischemia and reperfusion.
    Circulation 06/2014; · 15.20 Impact Factor
  • N. M. Htun, Karlheinz Peter
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    ABSTRACT: Potential biomarkers have been investigated using proteomic studies in a variety of diseases. Some biomarkers have central roles in both diagnosis and monitoring of various disorders in clinical medicine, such as troponins, brain natriuretic peptide, and C-reactive protein. Although several biomarkers have been suggested in human abdominal aortic aneurysm (AAA), reliable markers have been lacking. In this issue, Moxon et al. [Proteomics Clin Appl. 2014, XX: XX-XX] undertook a broad and systematic proteomic approach toward identification of biomarkers in a commonly used AAA mouse model (AAA created by angiotensin-II infusion). In this mouse model, apolipoprotein C1 and matrix metalloproteinase-9 were identified as novel biomarkers of stable AAA. This finding represents an important step forward, toward a clinically relevant role of biomarkers in AAA. This also encourages for further studies toward the identification of biomarkers (or their combinations) that can predict AAA progression and rupture, which would represent a major progress in AAA management and would establish an AAA biomarker as a much anticipated clinical tool.This article is protected by copyright. All rights reservedThis article is protected by copyright. All rights reserved
    PROTEOMICS - CLINICAL APPLICATIONS 06/2014; · 1.81 Impact Factor
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    ABSTRACT: Tissue damage in burn injury leads to a rapid increase of leukocytes and acute phase reactants. Plasma levels of C-reactive protein (CRP) rise within hours after the insult. No deficiency of this protein has been reported in humans, suggesting it plays a pivotal role in innate immunity. CRP in circulation is composed of five identical subunits (pCRP). Recently deposits of structurally modified CRP (mCRP) have been found in inflammatory diseases. Little is known about this structural change and how it affects CRP functions. We analyzed CRP deposits in burn wounds and serum by immunohistochemistry, Western blot and dot blot analysis. CRP was deposited in necrotic and inflamed tissue, but not in adjacent healthy tissue. Tissue deposited CRP was detected by mCRP-specific antibodies and structurally different from serum pCRP. mCRP but not pCRP induced reactive oxygen species production by monocytes and facilitated uptake of necrotic Jurkat cells by macrophages. In addition it accelerated migration of keratinocytes in a scratch wound assay. The structural changes that occur in pCRP upon localization to damaged and inflamed tissue in burn wounds result in a functionally altered protein with distinct functions. mCRP exhibits opsonic, pro-inflammatory and pro-migratory properties which modulate wound healing.
    International immunology. 05/2014;
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    ABSTRACT: The enzyme-mediated site-specific bioconjugation of a radioactive metal complex to a single-chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single-chain variable fragment. The antibody fragment scFvanti-LIBS targets ligand-induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron-emitting isotope 64Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site-specific enzyme-mediated conjugation and should be broadly applicable to other metal complexes and proteins.
    Angewandte Chemie 04/2014;
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The enzyme-mediated site-specific bioconjugation of a radioactive metal complex to a single-chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single-chain variable fragment. The antibody fragment scFvanti-LIBS targets ligand-induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron-emitting isotope 64Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site-specific enzyme-mediated conjugation and should be broadly applicable to other metal complexes and proteins.
    Angewandte Chemie International Edition 04/2014; · 11.34 Impact Factor
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    ABSTRACT: Metal–organic coordination materials are of widespread interest because of the coupled benefits of inorganic and organic building blocks. These materials can be assembled into hollow capsules with a range of properties, which include selective permeability, enhanced mechanical/thermal stability, and stimuli‐responsiveness. Previous studies have primarily focused on the assembly aspects of metal‐coordination capsules; however, the engineering of metal‐specific functionality for capsule design has not been explored. A library of functional metal–phenolic network (MPN) capsules prepared from a phenolic ligand (tannic acid) and a range of metals is reported. The properties of the MPN capsules are determined by the coordinated metals, allowing for control over film thickness, disassembly characteristics, and fluorescence behavior. Furthermore, the functional properties of the MPN capsules were tailored for drug delivery, positron emission tomography (PET), magnetic resonance imaging (MRI), and catalysis. The ability to incorporate multiple metals into MPN capsules demonstrates that a diverse range of functional materials can be generated. GermanMultifunktionelle Kapseln: Gerbsäure wurde verwendet, um eine Vielzahl von Metallen in einem einstufigen Selbstorganisationsprozess zu koordinieren und eine Bibliothek von Metall‐Phenol‐Netzwerken (MPNs; siehe Bild) aufzubauen. Die Eigenschaften der MPN‐Kapseln sind von den koordinierten Metallen bestimmt.
    Angewandte Chemie 04/2014;
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    [Show abstract] [Hide abstract]
    ABSTRACT: Metal–organic coordination materials are of widespread interest because of the coupled benefits of inorganic and organic building blocks. These materials can be assembled into hollow capsules with a range of properties, which include selective permeability, enhanced mechanical/thermal stability, and stimuli-responsiveness. Previous studies have primarily focused on the assembly aspects of metal-coordination capsules; however, the engineering of metal-specific functionality for capsule design has not been explored. A library of functional metal–phenolic network (MPN) capsules prepared from a phenolic ligand (tannic acid) and a range of metals is reported. The properties of the MPN capsules are determined by the coordinated metals, allowing for control over film thickness, disassembly characteristics, and fluorescence behavior. Furthermore, the functional properties of the MPN capsules were tailored for drug delivery, positron emission tomography (PET), magnetic resonance imaging (MRI), and catalysis. The ability to incorporate multiple metals into MPN capsules demonstrates that a diverse range of functional materials can be generated.
    Angewandte Chemie 04/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: Metal–organic coordination materials are of widespread interest because of the coupled benefits of inorganic and organic building blocks. These materials can be assembled into hollow capsules with a range of properties, which include selective permeability, enhanced mechanical/thermal stability, and stimuli-responsiveness. Previous studies have primarily focused on the assembly aspects of metal-coordination capsules; however, the engineering of metal-specific functionality for capsule design has not been explored. A library of functional metal–phenolic network (MPN) capsules prepared from a phenolic ligand (tannic acid) and a range of metals is reported. The properties of the MPN capsules are determined by the coordinated metals, allowing for control over film thickness, disassembly characteristics, and fluorescence behavior. Furthermore, the functional properties of the MPN capsules were tailored for drug delivery, positron emission tomography (PET), magnetic resonance imaging (MRI), and catalysis. The ability to incorporate multiple metals into MPN capsules demonstrates that a diverse range of functional materials can be generated.
    Angewandte Chemie International Edition 04/2014; · 11.34 Impact Factor
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    ABSTRACT: Co-stimulatory cascades such as the CD40L-CD40 dyad enhance immune cell activation and inflammation during atherosclerosis. Here, we tested the hypothesis that CD40 directly modulates traits of the metabolic syndrome in diet-induced obesity (DIO) in mice. To induce the metabolic syndrome, WT or CD40(-/-) mice consumed a high fat diet for 20 weeks. Unexpectedly, CD40(-/-) mice exhibited increased weight gain, impaired insulin secretion, augmented accumulation of inflammatory cells in adipose tissue, and enhanced pro-inflammatory gene expression. This pro-inflammatory and adverse metabolic phenotype could be transplanted into WT mice by reconstitution with CD40-deficient lymphocytes, indicating a major role for CD40 on T- or B cells in this context. Conversely, therapeutic activation of CD40 signaling by the stimulating antibody FGK45 abolished further weight gain during the study, lowered glucose levels, improved insulin sensitivity, and suppressed adipose tissue inflammation. Mechanistically, CD40 activation decreased expression of pro-inflammatory cytokines in T cells, but not in B cells or macrophages. Finally, re-population of lymphocyte-free Rag1(-/-) mice with CD40(-/-) T cells provoked dysmetabolism and inflammation, corroborating a protective role of CD40 on T cells in the metabolic syndrome. Finally, levels of sCD40 showed a positive association with obesity in humans, suggesting clinical relevance of our findings. We present the surprising finding that CD40 deficiency on T cells aggravates, while activation of CD40 signaling improves, adipose tissue inflammation and its metabolic complications. Positive modulation of the CD40 pathway might, therefore, describe a novel therapeutic concept against cardio-metabolic disease.
    Circulation 03/2014; · 15.20 Impact Factor
  • Global Heart. 03/2014; 9(1):e89.
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    ABSTRACT: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted leaving many patients untreated. We developed a novel targeted fibrinolytic drug that is directed against activated platelets. We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet-integrin GPIIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5-scuPA (75U/g BW) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared to saline (p<0.001) and blood flow recovery was similar to high dose non-targeted urokinase (500U/g BW). Tail bleeding time was significantly prolonged with this high dose of non-targeted urokinase, but not with equally effective targeted scFvSCE5-scuPA at 75U/g BW. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75U/g BW scFvSCE5-scuPA as compared to same dose of a mutated, non-targeting scFv-scuPA or vehicle. The ability of scFvSCE5-scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. Targeting of scuPA to activated GPIIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.
    Circulation Research 02/2014; · 11.86 Impact Factor
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    ABSTRACT: Atherosclerosis is driven by inflammatory reactions that are shared with the innate immune system. Toll-like receptor-9 (TLR9) is an intracellular pattern recognition receptor of the innate immune system that is currently under clinical investigation as a therapeutic target in inflammatory diseases. Here, we investigated whether TLR9 has a role in the development of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. Newly generated double-knockout ApoE(-/-):TLR9(-/-) mice and control ApoE(-/-) mice were fed a high-fat diet from 8 weeks and effects on lesion size, cellular composition, inflammatory status, and plasma lipids were assessed after 8, 12, 15, and 20 weeks. All 4 time points demonstrated exacerbated atherosclerotic lesion severity in ApoE(-/-):TLR9(-/-) mice, with a corresponding increase in lipid deposition and accumulation of macrophages, dendritic cells, and CD4(+) T cells. Although ApoE(-/-):TLR9(-/-) mice exhibited an increase in plasma very low-density lipoprotein/low-density-lipoprotein cholesterol, the very low-density lipoprotein/low-density lipoprotein:high-density lipoprotein ratio was unaltered because of a parallel increase in plasma high-density lipoprotein cholesterol. As a potential mechanism accounting for plaque progression in ApoE(-/-):TLR9(-/-) mice, CD4(+) T-cell accumulation was further investigated and depletion of these cells in ApoE(-/-):TLR9(-/-) mice significantly reduced lesion severity. As a final translational approach, administration of a TLR9 agonist (type B CpG oligodeoxynucleotide 1668) to ApoE(-/-) mice resulted in a reduction of lesion severity. Genetic deletion of the innate immune receptor TLR9 exacerbated atherosclerosis in ApoE(-/-) mice fed a high-fat diet. CD4(+) T cells were identified as potential mediators of this effect. A type B CpG oligodeoxynucleotide TLR9 agonist reduced lesion severity, thus identifying a novel therapeutic approach in atherosclerosis.
    Arteriosclerosis Thrombosis and Vascular Biology 01/2014; · 6.34 Impact Factor
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    ABSTRACT: Vascular thrombosis is a crucial event and still cause of significant morbidity and mortality worldwide. Deep vein thrombosis with subsequent pulmonary embolism constitutes a frequent clinical event, while reliable detection, especially of small or old thrombi, still remains clinically challenging. Occlusion or thromboembolism in an arterial vessel may result in myocardial infarction or stroke, and early detection would be of enormous clinical interest. Noninvasive molecular imaging techniques, particularly targeting key structures of developing or established thrombosis, have demonstrated its ability to detect this pathology in different models of disease, and current research is heading towards clinical translation. In this article, recent developments and challenges of molecular imaging of vascular thrombosis involving magnetic resonance imaging, ultrasound and nuclear imaging techniques are reviewed.
    Current Molecular Imaging. 01/2014; 3(1).
  • 09/2013;
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    ABSTRACT: Rationale: The high morbidity/mortality of atherosclerosis is typically precipitated by plaque rupture and consequent thrombosis. However, research on pathomechanisms and therapeutic approaches is limited by the lack of animal models that reproduce plaque instability as seen in humans. Objective: Development and utilization of a mouse model of plaque rupture that reflects the end stage of human atherosclerosis. Methods and Results: Based on flow measurements and computational fluid dynamics, we applied a tandem stenosis to the carotid artery of ApoE-/- mice on high fat diet. 7 weeks postoperatively, we observed in ~50% of mice disruption of fibrous caps, intraplaque hemorrhage, intra¬luminal thrombosis, neovascularization and other characteristics typically seen in humans. Administration of atorvastatin is associated with plaque stabilization and down regulation of MCP-1 and ubiquitin, as reported in humans. Microarray profiling demon¬strates major differences in the hierarchical clustering of genes and microRNAs between non-atherosclerotic arteries, stable and unstable plaques and allows the identification of distinct genes/microRNAs, potentially representing novel therapeutic targets for plaque stabilization. The use of the described animal model as a discovery tool was established in a pilot approach, identifying ADAMTS4 as a potential pathogenic factor in plaque instability in mice and humans. Conclusions: The newly described mouse model reflects human atherosclerotic plaque instability and represents a discovery tool towards the development and testing of therapeutic strategies aimed at preventing plaque rupture. Distinctly expressed genes and microRNAs can be linked to plaque instability. Key words: atherosclerosis; plaque instability; plaque rupture; mouse model; gene expression profiling; microRNA profiling
    Circulation Research 07/2013; 112(15). · 11.86 Impact Factor

Publication Stats

2k Citations
689.32 Total Impact Points

Institutions

  • 2007–2014
    • Baker IDI Heart and Diabetes Institute
      Melbourne, Victoria, Australia
    • Brigham and Women's Hospital
      • Center for Brain Mind Medicine
      Boston, MA, United States
  • 2013
    • Alfred Hospital
      Melbourne, Victoria, Australia
  • 2002–2013
    • University of Freiburg
      • • Internal Medicine 2 - Gastroenterology, Hepatology, Endocrinology and Infectious Diseases
      • • Department of Internal Medicine
      Freiburg, Baden-Württemberg, Germany
  • 2012
    • University of Melbourne
      • Department of Chemical and Biomolecular Engineering
      Melbourne, Victoria, Australia
    • Universitäts-Herzzentrum Freiburg - Bad Krozingen
      باد کروزینگن, Baden-Württemberg, Germany
  • 2008–2012
    • Universitätsklinikum Freiburg
      • • Department of Internal Medicine III
      • • Department of Plastic and Hand Surgery
      • • Department of Cardiology and Angiology II
      Freiburg an der Elbe, Lower Saxony, Germany
    • Heart Research Institute
      Newtown, New South Wales, Australia
  • 2009
    • Third Military Medical University
      Ch’ung-ch’ing-shih, Chongqing Shi, China
  • 2004
    • University of North Carolina at Chapel Hill
      North Carolina, United States