Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain... more 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 (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) 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-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar 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.
Nanoporous metal-phenolic particles are fabricated through the nanostructural replication of dens... more Nanoporous metal-phenolic particles are fabricated through the nanostructural replication of dense Fe(III) -TA complexes in nanoporous CaCO3 template particles. The particles have potential for the diagnostic detection of endogenous levels of H2 O2 ex vivo and in vivo by ultrasound imaging, which is based on the catalytic activity of the coordinated Fe(3+) in the particles to break down H2 O2 to O2 microbubbles.
Advanced materials (Deerfield Beach, Fla.), Jan 3, 2015
Smart poly(2-oxazoline) (POx)-based multifunctional polymer capsules that specifically target gly... more Smart poly(2-oxazoline) (POx)-based multifunctional polymer capsules that specifically target glycoprotein (GP) IIb/IIIa on the surface of activated platelets are degraded by the serine protease thrombin and release the urokinase plasminogen activator loaded into the polymer capsules, only in the area of acute thrombosis.
Dual-responsive boronate-phenolic network (BPN) capsules are fabricated by the complexation of ph... more Dual-responsive boronate-phenolic network (BPN) capsules are fabricated by the complexation of phenylborate and phenolic materials. The BPN capsules are stable in the presence of competing carbohydrates, but dissociate at acidic pH or in the presence of competing cis-diols at physiological pH. This engineered capsule system provides a platform for a wide range of biological and biomedical applications.
Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microp... more 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.
We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-termin... more We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv(59D8)). Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target. In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clot-bound and prothrombinase-associated fXa. Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications.TAP is a highly potent fXa inhibitor. Since its N-terminus is essential for anti-fXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule. Two step affinity chromatography with Ni(2+) and beta(15-22)-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity. The recombinant fusion did not destroy the function of the fusion partners. Antibody binding function was on a par with the parent molecule. TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency. In human whole blood clots, scFv(59D8)-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all. In summary, scFv(59D8)-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition. The production in E. coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.
Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain... more 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 (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) 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-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar 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.
Targeted delivery or "smart delivery" of pharma... more Targeted delivery or "smart delivery" of pharmaceutical or imaging agents and even entire cells such as stem cells is an emerging trend in modern biotechnology. A binding ligand such as an antibody that can specifically bind to receptors expressed at a disease site is an essential component of such constructs. Different chemical methods have been widely used to apply antibodies for delivery systems; however, they typically result in impairment or loss of antibody functionality. Enzyme-mediated conjugation approaches have been developed to overcome this major disadvantage of conventional chemical methods. Sortase, an enzyme derived from Staphylococcus aureus, is able to provide a biochemically robust, highly reproducible, and site-specific coupling method for the conjugation of antibodies to pharmaceutical agents, nanoparticles, and cells for drug delivery, molecular imaging, and cell homing. Here, we review the use of sortase and other enzyme-based methods as bioconjugation tools with a focus on cardiovascular applications.
Over three decades after the generation of the first mouse monoclonal antibodies by Kohler and Mi... more Over three decades after the generation of the first mouse monoclonal antibodies by Kohler and Milstein, recombinant antibodies are the fastest growing class of therapeutic proteins. Furthermore, antibodies are key detection reagents in research and diagnostics. Technology improvements have provided several approaches to manufacturing human antibodies with high affinity for biologically relevant targets. Approximately 300 development programs for therapeutic antibodies have been reported in industrial and academic laboratories, and this clearly demonstrates the expectations towards antibody technology. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as one of the smallest possible format for recombinant antibodies and their use as diagnostic tools and therapeutic agents. We describe the structure, selection and production of single-chain antibodies. Furthermore, we review current applications of antibody fragments focusing on thrombus targeting using fibrin- and platelet-specific single-chain antibodies as well as describing novel noninvasive imaging approaches for the diagnosis of thrombosis and inflammation.
Antibodies are the most rapidly growing class of human therapeutics and the second largest class ... more Antibodies are the most rapidly growing class of human therapeutics and the second largest class of drugs after vaccines. At present, several antibodies are approved for therapeutic use in diverse clinical settings, including oncology, chronic inflammatory diseases, transplantation, infectious diseases, and cardiovascular medicine. These approved antibody therapeutics include unmodified immunoglobulin G molecules, radioimmunoconjugates, antibody-drug conjugates, and fragment antigen-binding molecules. At least 150 additional antibodies are in clinical development. A major strength of therapeutic antibodies is their established properties as a drug class with high success rates from clinical trials to regulatory approvals. Much of the experience gained from the generation and optimization of one antibody is applicable to other antibodies. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as the smallest possible format for recombinant antibodies, and their use as antithrombotic drugs. We describe different antibody formats, the current applications of antibody fragments, and their generation by cloning from hybridoma cell lines as well as their selection from antibody libraries. We review the use of antibody fragments for thrombus targeting using fibrin and platelet-specific single-chain antibodies in combination with anticoagulants and thrombolytic agents as antithrombotic drugs.
Activated platelets are key players in thrombosis and inflammation. We previously generated singl... more Activated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel (18)F PET radiotracer based on this antibody. ScFv(anti-LIBS) and control antibody mut-scFv were reacted with N-succinimidyl-4-[(18)F]fluorobenzoate (S[(18)F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl(3) induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied. After incubation with increasing concentrations of (18)F-scFv(anti-LIBS) clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled (18)F-mut-scFv (13.3 ± 3.8 vs. 3.6 ± 1 KBq, p < 0.05, n = 9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6 ± 4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7 ± 0.9% ID/g in the non-injured vessel 5 minutes after injection (p < 0.05, n = 6). Our results show that the novel antibody radiotracer (18)F-scFv(anti-LIBS) is useful for the sensitive detection of activated platelets and thrombosis. We describe the first (18)F variant of a scFv(anti-LIBS) against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future.
Several cytochrome P450 subfamilies are inducible by specific exogenous compounds like the antiep... more Several cytochrome P450 subfamilies are inducible by specific exogenous compounds like the antiepileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones also contributing to neuronal differentiation. CYP3A enzymes have the capacity to catalyze the hydroxylation of testosterone and a wide variety of therapeutic agents, but little is known about the expression and potential function of this subfamily in mouse brain. Here, we report the identification of mouse CYP3A isoforms, their induction and localization in mouse brain. Western blot analysis with anti-CYP3A1 antibodies revealed the phenytoin-inducible expression of CYP3A in brain microsomes, and also a constitutive expression of members of this subfamily in brain mitochondria. Using RT-PCR with a consensus primer pair for known mouse liver CYP3A-isoforms we could demonstrate the expression of CYP3A11 and 3A13 mRNA in mouse brain. Finally, using double immunofluorescence labeling we analyzed the histoanatomical distribution of CYP3A throughout the brain with confocal laser scanning microscopy. We found strong immunoreactivity in neurons of hippocampus and hypothalamic areas which are sensitive to steroid hormones. CYP3A immunoreactivity was apparent also in neurons of the cerebellum, the thalamus and the olfactory bulb. Non-neuronal expression of CYP3A could be found in some astrocyte populations and in vascular as well as ventricular border lines. The presence of CYP3A predominantly in neurons but also in cells contributing to the blood-brain and blood-liquor barrier suggests important roles of this subfamily in mediation of steroid hormone action in mouse brain as well as in preventing the brain from potentially cytotoxic compounds.
Steroid hormones influence the development of undifferentiated brain during ontogenesis. In the p... more Steroid hormones influence the development of undifferentiated brain during ontogenesis. In the present study we investigated the metabolic pathway of testosterone in immortalized embryonic and postnatal hippocampal neurons from C57BL/6 mice. Both cell lines are capable of metabolizing testosterone to 6alpha-hydroxytestosterone, 6beta-hydroxytestosterone and androstenedione. The formation was found to correlate with protein concentration and time of incubation. These linearities were significant for all metabolites except androstenedione that was the main metabolite in embryonic hippocampal neurons and nearly absent in postnatal neurons. Moreover, only embryonic cells react to testosterone with a decrease of beta-tubulin expression, that was a typical effect indicating induced neuronal maturation. Application of androstenedione caused the same decrease of beta-tubulin expression as testosterone did before. Our results of hippocampal testosterone metabolism in vitro confirm that not only estradiol and 5alpha-dihydrotestosterone could impact neural tissue but also androstenedione is a powerful metabolite involved in prenatal neuronal differentiation.
Epilepsy is very often related to strong impairment of neuronal networks, particularly in the hip... more Epilepsy is very often related to strong impairment of neuronal networks, particularly in the hippocampus. Previous studies of brain tissue have demonstrated that long-term administration of the anti-epileptic drug (AED) phenytoin leads to enhanced metabolism of testosterone mediated by cytochrome P450 (CYP) isoforms. Thus, we speculate that AEDs affect androgen signalling in the hippocampus. In the present study, we investigated how the AED phenytoin influences the levels of testosterone, 17beta-oestradiol, and androgen receptor (AR) in the hippocampus of male C57Bl/6J mice. Phenytoin administration led to a 61.24% decreased hippocampal testosterone level as compared with controls, while serum levels were slightly enhanced. 17beta-Oestradiol serum level was elevated 2.6-fold. Concomitantly, the testosterone metabolizing CYP isoforms CYP3A11 and CYP19 (aromatase) have been found to be induced 2.4- and 4.2-fold, respectively. CYP3A-mediated depletion of testosterone-forming 2beta-, and 6beta-hydroxytestosterone was significantly enhanced. Additionally, AR expression was increased 2-fold (mRNA) and 1.8-fold (protein), predominantly in the CA1 region. AR was shown to concentrate in nuclei of CA1 pyramidal neurons. We conclude that phenytoin affects testosterone metabolism via induction of CYP isoforms. The increased metabolism of testosterone leading to augmented androgen metabolite formation most likely led to enhanced expression of CYP19 and AR in hippocampus. Phenytoin obviously modulates the androgen signalling in the hippocampus.
Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different area... more Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.
In the hippocampus, the synaptic vesicle protein synaptoporin (SPO) has been reported to be exclu... more In the hippocampus, the synaptic vesicle protein synaptoporin (SPO) has been reported to be exclusively enriched in the granule cell axons, the mossy fibers. In this study, we show that in adult rats and mice SPO immunoreactivity (IR) is also detectable in strata oriens, radiatum, and lacunosum-moleculare of CA1-CA3, as well as perisomatically in the hippocampus proper and fascia dentata. In situ hybridization confirmed that SPO mRNA was present in granule cells and CA3 pyramidal cells but not in CA1 pyramidal cells. Importantly, cells scattered throughout the hippocampal layers resembling the distribution of interneurons were found to synthesize high amounts of SPO mRNA, too. Thus, these findings indicate that SPO expression in the hippocampus was underestimated until now. Moreover, double-labeling immunohistochemistry and confocal microscopy revealed selective colocalization of SPO and glutamate decarboxylase (GAD 65), a marker for gamma-aminobutyric acid (GABA)ergic terminals. To identify SPO expressing interneurons, in situ hybridization was combined with immunocytochemistry against parvalbumin (PV), calbindin (CB), calretinin (CR), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP). We found that SPO transcripts were differentially expressed by various interneuron subpopulations in the hippocampus of C57Bl/6 mice (PV 44.2%, CB 46.3%, CR 19.3%, CCK 38.6%, VIP 59.9%). Immunoelectron microscopy for SPO labeled synaptic vesicle profiles in distinct symmetric and asymmetric synapses. In conclusion, our data demonstrate that hippocampal principal cells and interneurons display a variety of synaptic vesicles that are likely to contribute to the functional characteristics of their output synapses.
Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain... more 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 (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) 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-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar 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.
Nanoporous metal-phenolic particles are fabricated through the nanostructural replication of dens... more Nanoporous metal-phenolic particles are fabricated through the nanostructural replication of dense Fe(III) -TA complexes in nanoporous CaCO3 template particles. The particles have potential for the diagnostic detection of endogenous levels of H2 O2 ex vivo and in vivo by ultrasound imaging, which is based on the catalytic activity of the coordinated Fe(3+) in the particles to break down H2 O2 to O2 microbubbles.
Advanced materials (Deerfield Beach, Fla.), Jan 3, 2015
Smart poly(2-oxazoline) (POx)-based multifunctional polymer capsules that specifically target gly... more Smart poly(2-oxazoline) (POx)-based multifunctional polymer capsules that specifically target glycoprotein (GP) IIb/IIIa on the surface of activated platelets are degraded by the serine protease thrombin and release the urokinase plasminogen activator loaded into the polymer capsules, only in the area of acute thrombosis.
Dual-responsive boronate-phenolic network (BPN) capsules are fabricated by the complexation of ph... more Dual-responsive boronate-phenolic network (BPN) capsules are fabricated by the complexation of phenylborate and phenolic materials. The BPN capsules are stable in the presence of competing carbohydrates, but dissociate at acidic pH or in the presence of competing cis-diols at physiological pH. This engineered capsule system provides a platform for a wide range of biological and biomedical applications.
Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microp... more 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.
We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-termin... more We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv(59D8)). Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target. In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clot-bound and prothrombinase-associated fXa. Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications.TAP is a highly potent fXa inhibitor. Since its N-terminus is essential for anti-fXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule. Two step affinity chromatography with Ni(2+) and beta(15-22)-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity. The recombinant fusion did not destroy the function of the fusion partners. Antibody binding function was on a par with the parent molecule. TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency. In human whole blood clots, scFv(59D8)-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all. In summary, scFv(59D8)-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition. The production in E. coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.
Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain... more 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 (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) 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-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar 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.
Targeted delivery or "smart delivery" of pharma... more Targeted delivery or "smart delivery" of pharmaceutical or imaging agents and even entire cells such as stem cells is an emerging trend in modern biotechnology. A binding ligand such as an antibody that can specifically bind to receptors expressed at a disease site is an essential component of such constructs. Different chemical methods have been widely used to apply antibodies for delivery systems; however, they typically result in impairment or loss of antibody functionality. Enzyme-mediated conjugation approaches have been developed to overcome this major disadvantage of conventional chemical methods. Sortase, an enzyme derived from Staphylococcus aureus, is able to provide a biochemically robust, highly reproducible, and site-specific coupling method for the conjugation of antibodies to pharmaceutical agents, nanoparticles, and cells for drug delivery, molecular imaging, and cell homing. Here, we review the use of sortase and other enzyme-based methods as bioconjugation tools with a focus on cardiovascular applications.
Over three decades after the generation of the first mouse monoclonal antibodies by Kohler and Mi... more Over three decades after the generation of the first mouse monoclonal antibodies by Kohler and Milstein, recombinant antibodies are the fastest growing class of therapeutic proteins. Furthermore, antibodies are key detection reagents in research and diagnostics. Technology improvements have provided several approaches to manufacturing human antibodies with high affinity for biologically relevant targets. Approximately 300 development programs for therapeutic antibodies have been reported in industrial and academic laboratories, and this clearly demonstrates the expectations towards antibody technology. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as one of the smallest possible format for recombinant antibodies and their use as diagnostic tools and therapeutic agents. We describe the structure, selection and production of single-chain antibodies. Furthermore, we review current applications of antibody fragments focusing on thrombus targeting using fibrin- and platelet-specific single-chain antibodies as well as describing novel noninvasive imaging approaches for the diagnosis of thrombosis and inflammation.
Antibodies are the most rapidly growing class of human therapeutics and the second largest class ... more Antibodies are the most rapidly growing class of human therapeutics and the second largest class of drugs after vaccines. At present, several antibodies are approved for therapeutic use in diverse clinical settings, including oncology, chronic inflammatory diseases, transplantation, infectious diseases, and cardiovascular medicine. These approved antibody therapeutics include unmodified immunoglobulin G molecules, radioimmunoconjugates, antibody-drug conjugates, and fragment antigen-binding molecules. At least 150 additional antibodies are in clinical development. A major strength of therapeutic antibodies is their established properties as a drug class with high success rates from clinical trials to regulatory approvals. Much of the experience gained from the generation and optimization of one antibody is applicable to other antibodies. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as the smallest possible format for recombinant antibodies, and their use as antithrombotic drugs. We describe different antibody formats, the current applications of antibody fragments, and their generation by cloning from hybridoma cell lines as well as their selection from antibody libraries. We review the use of antibody fragments for thrombus targeting using fibrin and platelet-specific single-chain antibodies in combination with anticoagulants and thrombolytic agents as antithrombotic drugs.
Activated platelets are key players in thrombosis and inflammation. We previously generated singl... more Activated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel (18)F PET radiotracer based on this antibody. ScFv(anti-LIBS) and control antibody mut-scFv were reacted with N-succinimidyl-4-[(18)F]fluorobenzoate (S[(18)F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl(3) induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied. After incubation with increasing concentrations of (18)F-scFv(anti-LIBS) clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled (18)F-mut-scFv (13.3 ± 3.8 vs. 3.6 ± 1 KBq, p < 0.05, n = 9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6 ± 4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7 ± 0.9% ID/g in the non-injured vessel 5 minutes after injection (p < 0.05, n = 6). Our results show that the novel antibody radiotracer (18)F-scFv(anti-LIBS) is useful for the sensitive detection of activated platelets and thrombosis. We describe the first (18)F variant of a scFv(anti-LIBS) against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future.
Several cytochrome P450 subfamilies are inducible by specific exogenous compounds like the antiep... more Several cytochrome P450 subfamilies are inducible by specific exogenous compounds like the antiepileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones also contributing to neuronal differentiation. CYP3A enzymes have the capacity to catalyze the hydroxylation of testosterone and a wide variety of therapeutic agents, but little is known about the expression and potential function of this subfamily in mouse brain. Here, we report the identification of mouse CYP3A isoforms, their induction and localization in mouse brain. Western blot analysis with anti-CYP3A1 antibodies revealed the phenytoin-inducible expression of CYP3A in brain microsomes, and also a constitutive expression of members of this subfamily in brain mitochondria. Using RT-PCR with a consensus primer pair for known mouse liver CYP3A-isoforms we could demonstrate the expression of CYP3A11 and 3A13 mRNA in mouse brain. Finally, using double immunofluorescence labeling we analyzed the histoanatomical distribution of CYP3A throughout the brain with confocal laser scanning microscopy. We found strong immunoreactivity in neurons of hippocampus and hypothalamic areas which are sensitive to steroid hormones. CYP3A immunoreactivity was apparent also in neurons of the cerebellum, the thalamus and the olfactory bulb. Non-neuronal expression of CYP3A could be found in some astrocyte populations and in vascular as well as ventricular border lines. The presence of CYP3A predominantly in neurons but also in cells contributing to the blood-brain and blood-liquor barrier suggests important roles of this subfamily in mediation of steroid hormone action in mouse brain as well as in preventing the brain from potentially cytotoxic compounds.
Steroid hormones influence the development of undifferentiated brain during ontogenesis. In the p... more Steroid hormones influence the development of undifferentiated brain during ontogenesis. In the present study we investigated the metabolic pathway of testosterone in immortalized embryonic and postnatal hippocampal neurons from C57BL/6 mice. Both cell lines are capable of metabolizing testosterone to 6alpha-hydroxytestosterone, 6beta-hydroxytestosterone and androstenedione. The formation was found to correlate with protein concentration and time of incubation. These linearities were significant for all metabolites except androstenedione that was the main metabolite in embryonic hippocampal neurons and nearly absent in postnatal neurons. Moreover, only embryonic cells react to testosterone with a decrease of beta-tubulin expression, that was a typical effect indicating induced neuronal maturation. Application of androstenedione caused the same decrease of beta-tubulin expression as testosterone did before. Our results of hippocampal testosterone metabolism in vitro confirm that not only estradiol and 5alpha-dihydrotestosterone could impact neural tissue but also androstenedione is a powerful metabolite involved in prenatal neuronal differentiation.
Epilepsy is very often related to strong impairment of neuronal networks, particularly in the hip... more Epilepsy is very often related to strong impairment of neuronal networks, particularly in the hippocampus. Previous studies of brain tissue have demonstrated that long-term administration of the anti-epileptic drug (AED) phenytoin leads to enhanced metabolism of testosterone mediated by cytochrome P450 (CYP) isoforms. Thus, we speculate that AEDs affect androgen signalling in the hippocampus. In the present study, we investigated how the AED phenytoin influences the levels of testosterone, 17beta-oestradiol, and androgen receptor (AR) in the hippocampus of male C57Bl/6J mice. Phenytoin administration led to a 61.24% decreased hippocampal testosterone level as compared with controls, while serum levels were slightly enhanced. 17beta-Oestradiol serum level was elevated 2.6-fold. Concomitantly, the testosterone metabolizing CYP isoforms CYP3A11 and CYP19 (aromatase) have been found to be induced 2.4- and 4.2-fold, respectively. CYP3A-mediated depletion of testosterone-forming 2beta-, and 6beta-hydroxytestosterone was significantly enhanced. Additionally, AR expression was increased 2-fold (mRNA) and 1.8-fold (protein), predominantly in the CA1 region. AR was shown to concentrate in nuclei of CA1 pyramidal neurons. We conclude that phenytoin affects testosterone metabolism via induction of CYP isoforms. The increased metabolism of testosterone leading to augmented androgen metabolite formation most likely led to enhanced expression of CYP19 and AR in hippocampus. Phenytoin obviously modulates the androgen signalling in the hippocampus.
Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different area... more Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.
In the hippocampus, the synaptic vesicle protein synaptoporin (SPO) has been reported to be exclu... more In the hippocampus, the synaptic vesicle protein synaptoporin (SPO) has been reported to be exclusively enriched in the granule cell axons, the mossy fibers. In this study, we show that in adult rats and mice SPO immunoreactivity (IR) is also detectable in strata oriens, radiatum, and lacunosum-moleculare of CA1-CA3, as well as perisomatically in the hippocampus proper and fascia dentata. In situ hybridization confirmed that SPO mRNA was present in granule cells and CA3 pyramidal cells but not in CA1 pyramidal cells. Importantly, cells scattered throughout the hippocampal layers resembling the distribution of interneurons were found to synthesize high amounts of SPO mRNA, too. Thus, these findings indicate that SPO expression in the hippocampus was underestimated until now. Moreover, double-labeling immunohistochemistry and confocal microscopy revealed selective colocalization of SPO and glutamate decarboxylase (GAD 65), a marker for gamma-aminobutyric acid (GABA)ergic terminals. To identify SPO expressing interneurons, in situ hybridization was combined with immunocytochemistry against parvalbumin (PV), calbindin (CB), calretinin (CR), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP). We found that SPO transcripts were differentially expressed by various interneuron subpopulations in the hippocampus of C57Bl/6 mice (PV 44.2%, CB 46.3%, CR 19.3%, CCK 38.6%, VIP 59.9%). Immunoelectron microscopy for SPO labeled synaptic vesicle profiles in distinct symmetric and asymmetric synapses. In conclusion, our data demonstrate that hippocampal principal cells and interneurons display a variety of synaptic vesicles that are likely to contribute to the functional characteristics of their output synapses.
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Papers by Christoph E Hagemeyer