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Thrombolysis with tissue-type plasminogen activator (tPA) remains the main treatment for acute ischemic stroke. Nevertheless, tPA intervention is limited by a short therapeutic window, low recanalization rates, and a risk of intracranial hemorrhage (ICH), highlighting the clinical demand for improved thrombolytic drugs. We examined a novel thrombolytic agent termed “SCE5-scuPA,” comprising a single-chain urokinase plasminogen activator (scuPA) fused with a single-chain antibody (SCE5) that targets the activated glycoprotein IIb/IIIa platelet receptor, for its effects in experimental stroke. SCE5-scuPA was first tested in a whole blood clot degradation assay to show the benefit of platelet-targeted thrombolysis. The tail bleeding time, blood clearance, and biodistribution were then determined to inform the use of SCE5-scuPA in mouse models of photothrombotic stroke and middle cerebral artery occlusion against tenecteplase. The impacts of SCE5-scuPA on motor function, ICH, blood–brain barrier (BBB) integrity, and immunosuppression were evaluated. Infarct size was measured by computed tomography imaging and magnetic resonance imaging. SCE5-scuPA enhanced clot degradation ex vivo compared with its nonplatelet-targeting control. The maximal SCE5-scuPA dose that maintained hemostasis and a rapid blood clearance was determined. SCE5-scuPA administration both before and 2 hours after photothrombotic stroke reduced the infarct volume. SCE5-scuPA also improved neurologic deficit, decreased intracerebral blood deposits, preserved the BBB, and alleviated immunosuppression poststroke. In middle cerebral artery occlusion, SCE5-scuPA did not worsen stroke outcomes or cause ICH, and it protected the BBB. Our findings support the ongoing development of platelet-targeted thrombolysis with SCE5-scuPA as a novel emergency treatment for acute ischemic stroke with a promising safety profile.

Glucose-responsive insulin-delivery platforms that are sensitive to dynamic glucose concentration fluctuations and provide both rapid and prolonged insulin release have great potential to control hyperglycemia and avoid hypoglycemia diabetes. Here, biodegradable and charge-switchable phytoglycogen nanoparticles capable of glucose-stimulated insulin release are engineered. The nanoparticles are “nanosugars” bearing glucose-sensitive phenylboronic acid groups and amine moieties that allow effective complexation with insulin (≈95% loading capacity) to form nanocomplexes. A single subcutaneous injection of nanocomplexes shows a rapid and efficient response to a glucose challenge in two distinct diabetic mouse models, resulting in optimal blood glucose levels (below 200 mg dL–1) for up to 13 h. The morphology of the nanocomplexes is found to be key to controlling rapid and extended glucoseregulated insulin delivery in vivo. These studies reveal that the injected nanocomplexes enabled efficient insulin release in the mouse, with optimal bioavailability, pharmacokinetics, and safety profiles. These results highlight a promising strategy for the development of a glucose-responsive insulin delivery system based on a natural and biodegradable nanosugar.

A highly effective 2-step system for site-specific antibody modification and conjugation of the monoclonal antibody Herceptin (commercially available under Trastuzumab) in a cysteine-independent manner was used to generate labelled antibodies for in vivo imaging. The first step contains redox-activated chemical tagging (ReACT) of thioethers via engineered methionine residues to introduce specific alkyne moieties, thereby offering a novel easy way to fundamentally change the process of antibody bioconjugation. The second step involves modification of the introduced alkyne via azide-alkyne cycloaddition 'click' conjugation. The versatility of this 2-step approach is demonstrated here by the selective incorporation of a fluorescent dye but can also be applied to a wide variety of different conjugation partners depending on the desired application in a facile manner. Methionine-modified antibodies were characterised in vitro, and the diagnostic potential of the most promising variant was further analysed in an in vivo xenograft animal model using a fluorescence imaging modality. This study demonstrates how methionine-mediated antibody conjugation offers an orthogonal and versatile route to the generation of tailored antibody conjugates with in vivo applicability.

Nisin, a peptide used as a natural food preservative, is employed in this work for the development of a novel nanocarrier system. Stable and uniform nisin-shelled nanoemulsions (NSNE) with a diameter of 100 ± 20 nm were successfully prepared using 20 kHz flow-through ultrasonication technique. The NSNE showed limited toxicity, high bactericidal activity and high drug loading capacity (EE 65 % w/w). In addition, the nisin shell was exploited for the site-specific attachment of a recombinantly produced cancer targeting ligand (αHER2

The morphology of nanomaterials critically influences their biological interactions. However, there is currently a lack of robust methods for preparing non-spherical particles from biocompatible materials. Here, we combine ‘living’ crystallisation-driven self-assembly (CDSA), a seeded growth method that enables the preparation of rod-like polymer nanoparticles, with poly(2-oxazoline)s (POx), a polymer class that exhibits ‘stealth’ behaviour and excellent biocompatibility. For the first time, the ‘living’ CDSA process was carried out in pure water, resulting in POx nanorods with lengths ranging from ∼60 to 635 nm. In vitro and in vivo study revealed low immune cell association and encouraging blood circulation times, but little difference in the behaviour of POx nanorods of different length. The stealth behaviour observed highlights the promising potential of POx nanorods as a next generation stealth drug delivery platform., Triggered by heating, a poly(2-alkyl-2-oxazoline) block copolymer undergoes seeded growth in water forming length tuneable nanorods. Morphology and composition combine to impart low immune cell association and promising blood circulation lifetimes.

We report the development of a theranostic collagen targeted cell penetrating drug delivery system toward treatment of cardiovascular disease. Caused by the action of matrix metalloproteinases (MMP...

Antibody (Ab)-targeted nanoparticles are becoming increasingly important for precision medicine. By controlling the Ab orientation, targeting properties can be enhanced; however, to afford such an ordered configuration, cumbersome chemical functionalization protocols are usually required. This aspect limits the progress of Abs-nanoparticles toward nanomedicine translation. Herein, a novel one-step synthesis of oriented monoclonal Ab-decorated metal-organic framework (MOF) nanocrystals is presented. The crystallization of a zinc-based MOF, Zn2 (mIM)2 (CO3 ), from a solution of Zn2+ and 2-methylimidazole (mIM), is triggered by the fragment crystallizable (Fc) region of the Ab. This selective growth yields biocomposites with oriented Abs on the MOF nanocrystals (MOF*Ab): the Fc regions are partially inserted within the MOF surface and the antibody-binding regions protrude from the MOF surface toward the target. This ordered configuration imparts antibody-antigen recognition properties to the biocomposite and shows preserved target binding when compared to the parental antibodies. Next, the biosensing performance of the system is tested by loading MOF*Ab with luminescent quantum dots (QD). The targeting efficiency of the QD-containing MOF*Ab is again, fully preserved. The present work represents a simple self-assembly approach for the fabrication of antibody-decorated MOF nanocrystals with broad potential for sensing, diagnostic imaging, and targeted drug delivery. Published version This work is supported by the National Health and Medical Research Council of Australia (Career Development Fellowship GNT1140465 to K.A., Senior Research Fellowship to C.E.H. GNT1154270).

Mentorship is a fundamental aspect that contributes to the success of a career in science, technology, engineering, and mathematics (STEM), particularly in academia. Research suggests that underrepresented minorities (URMs) often experience less quality mentorship and face barriers to finding successful mentor-mentee relationships. URM trainees in STEM face challenges that are not encountered by their majority peers or mentors, adding another level of complexity to establishing important relationships. Mentors of URM trainees must therefore mentor beyond general scientific training and tailor their mentorship to be more culturally appropriate and inclusive, allowing URM trainees to bring their whole selves to the table and leading to their effective socialization. Herein, we present the perspectives of group leaders and trainees from around the globe to highlight key aspects of creating successful mentor-mentee relationships that are sustainable and productive for both parties.

Drug carriers typically require both stealth and targeting properties to minimize nonspecific interactions with healthy cells and increase specific interaction with diseased cells. Herein, the assembly of targeted poly(ethylene glycol) (PEG) particles functionalized with cyclic peptides containing Arg-Gly-Asp (RGD) (ligand) using a mesoporous silica templating method is reported. The influence of PEG molecular weight, ligand-to-PEG molecule ratio, and particle size on cancer cell targeting to balance stealth and targeting of the engineered PEG particles is investigated. RGD-functionalized PEG particles (PEG-RGD particles) efficiently target U-87 MG cancer cells under static and flow conditions in vitro, whereas PEG and cyclic peptides containing Arg-Asp-Gly (RDG)-functionalized PEG (PEG-RDG) particles display negligible interaction with the same cells. Increasing the ligand-to-PEG molecule ratio improves cell targeting. In addition, the targeted PEG-RGD particles improve cell uptake via receptor-mediated endocytosis, which is desirable for intracellular drug delivery. The PEG-RGD particles show improved tumor targeting (14% ID g-1) when compared with the PEG (3% ID g-1) and PEG-RDG (7% ID g-1) particles in vivo, although the PEG-RGD particles show comparatively higher spleen and liver accumulation. The targeted PEG particles represent a platform for developing particles aimed at balancing nonspecific and specific interactions in biological systems.

Background Cardiac fibrosis is the excessive deposition of extracellular matrix in the heart, triggered by a cardiac insult, aging, genetics, or environmental factors. Molecular imaging of the cardiac extracellular matrix with targeted probes could improve diagnosis and treatment of heart disease. However, although this technology has been used to demonstrate focal scarring arising from myocardial infarction, its capacity to demonstrate extracellular matrix expansion and diffuse cardiac fibrosis has not been assessed. Methods and Results Here, we report the use of collagen‐targeted peptides labeled with near‐infrared fluorophores for the detection of diffuse cardiac fibrosis in the β2‐AR (β‐2‐adrenergic receptor) overexpressing mouse model and in ischemic human hearts. Two approaches were evaluated, the first based on a T peptide that binds matrix metalloproteinase‐2‐proteolyzed collagen IV, and the second on the cyclic peptide EP‐3533, which targets collagen I. The systemic and cardiac uptakes of both peptides (intravenously administered) were quantified ex vivo by near‐infrared imaging of whole organs, tissue sections, and heart lysates. The peptide accumulation profiles corresponded to an immunohistochemically‐validated increase in collagen types I and IV in hearts of transgenic mice versus littermate controls. The T peptide could encouragingly demonstrate both the intermediate (7 months old) and severe (11 months old) cardiomyopathic phenotypes. Co‐immunostainings of fluorescent peptides and collagens, as well as reduced collagen binding of a control peptide, confirmed the collagen specificity of the tracers. Qualitative analysis of heart samples from patients with ischemic cardiomyopathy compared with nondiseased donors supported the collagen‐enhancement capabilities of these peptides also in the clinical settings. Conclusions Together, these observations demonstrate the feasibility and translation potential of molecular imaging with collagen‐binding peptides for noninvasive imaging of diffuse cardiac fibrosis.

Background: A research priority in finding a cure for HIV is to establish methods to accurately locate and quantify where and how HIV persists in people living with HIV (PLWH) receiving suppressive antiretroviral therapy (ART). Infusing copper-64 (64Cu) radiolabelled broadly neutralising antibodies targeting HIV envelope (Env) with CT scan and positron emission tomography (PET) identified HIV Env in tissues in SIV infected non-human primates . We aimed to determine if a similar approach was effective in people living with HIV (PLWH). Methods: Unmodified 3BNC117 was compared with 3BNC117 bound to the chelator MeCOSar and 64Cu (64Cu-3BNC117) in vitro to assess binding and neutralization. In a clinical trial 64Cu-3BNC117 was infused into HIV uninfected (Group 1), HIV infected and viremic (viral load, VL >1000 c/mL; Group 2) and HIV infected aviremic (VL

Magnetic resonance imaging (MRI) is a powerful and indispensable tool in medical research, clinical diagnosis, and patient care due to its high spatial resolution and non-limited penetration depth. The simultaneous use of positive and negative MRI imaging that employs the same contrast agents will significantly improve detection accuracy. Here we report the development of functional multimodal iron oxide nanoparticles for targeted MRI of atherothrombosis using a combination of chemical and biological conjugation techniques. Monodisperse, water-soluble and biocompatible ultra-small magnetic dual contrast iron oxide nanoparticles (DCIONs) were generated using a high-temperature co-precipitation route and appeared to be efficient positive and negative dual contrast agents for magnetic resonance imaging. Using a unique chemo-enzymatic approach involving copper-free click chemistry and Staphylococcus aureus sortase A enzyme conjugation, DCIONs were functionalized with single-chain antibodies (scFv) directed against activated platelets for targeting purposes. The DCIONs were also labelled with fluorescent molecules to allow for optical imaging. The antigen binding activity of the scFv was retained and resulted in the successful targeting of contrast agents to thrombosis as demonstrated in a range of in vitro and in vivo experiments. T1- and T2-weighted MRI of thrombi was recorded and demonstrated the great potential of dual T1/T2 contrast iron oxide particles in imaging of cardiovascular disease.

Essentials: Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies. Summary: Background: Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives: Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods: PC-based nanocapsules (2.8 × 1012) with high-dose encapsulated eptifibatide were introduced into microfluidic blood perfusion assays and into in vivo models of thrombosis and tail bleeding. Results: Shear-triggered nanocapsule delivery of eptifibatide inhibited in vitro thrombus formation selectively under stenotic and high shear flow conditions above a shear rate of 1000 s-1 while leaving thrombus formation under physiologic shear rates unaffected. Thrombosis was effectively prevented in in vivo models of vessel wall damage. Importantly, mice infused with shear-sensitive antiplatelet nanocapsules did not show prolonged bleeding times. Conclusions: Targeted delivery of eptifibatide by shear-sensitive nanocapsules offers site-specific antiplatelet potential, and may form a basis for developing more potent and safer antiplatelet drugs.

The current advances in nanoengineered materials coupled with the precise targeting capability of recombinant antibodies can create nanoscale diagnostics and therapeutics which show enhanced accumulation and extended retention at a target tissue. Smaller antibodies such as single-chain variable fragments (scFv) preserve the selective and strong binding of their parent antibody to their antigen with the benefits of low immunogenicity, more efficient tissue penetration and easy introduction of functional residues suitable for site-specific conjugation. This is of high importance as nonspecific antibody modification often involves attachment to free cysteine or lysine amino acids which may reside in the active site, leading to reduced antigen binding.In this chapter, we outline a facile and versatile chemoenzymatic approach for production of targeted nanocarrier scFv conjugates using the bacterial trans-peptidase Sortase A (Srt A). Srt A efficiently mediates sequence-specific peptide ligation under mild conditions and has few undesirable side reactions. We first describe the production, purification and characterization of Srt A enzyme and a scFv construct which targets activated platelets, called scFv

MOFs have demonstrated outstanding properties for the protection and controlled release of different bio-entities, from proteins to living cells. Carbohydrates, as pure molecules or as a component of proteins and cells, perform essential biological functions. Thus, an understanding of the role of carbohydrates in the formation of MOF-based bio-composites will facilitate their application to biotechnology and medicine. Here, we investigate the role of carbohydrate molecular weight and chemical functionalization in the formation of carbohydrate@MOF composites. We find that chemical functionalization, such as carboxylation, that leads to an enhancement of metal cation concentration at the surface of the molecule triggers the rapid self-assembly of the MOF material, zeolitic-imidazolate framework 8 (ZIF-8). Furthermore, we determine the encapsulation efficiency and measure the release properties of the carbohydrate under controlled conditions. Our findings show that MOFs can be used to prepare a new class of biocomposites for the delivery of carbohydrate-based therapeutics.

The current advances in nanoengineered materials coupled with the precise targeting capability of recombinant antibodies can create nanoscale diagnostics and therapeutics which show enhanced accumulation and extended retention at a target tissue. Smaller antibodies such as single-chain variable fragments (scFv) preserve the selective and strong binding of their parent antibody to their antigen with the benefits of low immunogenicity, more efficient tissue penetration and easy introduction of functional residues suitable for site-specific conjugation. This is of high importance as nonspecific antibody modification often involves attachment to free cysteine or lysine amino acids which may reside in the active site, leading to reduced antigen binding.In this chapter, we outline a facile and versatile chemoenzymatic approach for production of targeted nanocarrier scFv conjugates using the bacterial trans-peptidase Sortase A (Srt A). Srt A efficiently mediates sequence-specific peptide ligation under mild conditions and has few undesirable side reactions. We first describe the production, purification and characterization of Srt A enzyme and a scFv construct which targets activated platelets, called scFvanti-GPIIb/IIIa. Following this, our protocol illustrates the chemoenzymatic modification of the antibody at the C-terminus with an orthogonal click chemistry linker. This avoids any random attachment to the biologically active antigen binding site of the antibody. Finally, we describe the modification of a nanoparticle surface with scFv attachment via two methods: (1) direct Sortase-mediated conjugation; or (2) a two-step system which consists of scFv Sortase-mediated conjugation followed by strain promoted azide-alkyne cycloaddition. Finally, methodology is described to assess the successful assembly of targeted particles.

MOFs have demonstrated outstanding properties for the protection and controlled release of different bio-entities, from proteins to living cells.

Brush polymers are highly functional polymeric materials combining the properties of different polymer classes and have found numerous applications, for example, in nanomedicine. Here, the synthesis of functional phosphonate‐ester‐bearing brush polymers based on poly(2‐oxazine)s is reported through a combination of cationic ring‐opening polymerization (CROP) of 2‐ethyl‐2‐oxazine and reversible addition‐fragmentation chain transfer (RAFT) polymerization. In this way, a small library of well‐defined (Đ ≤ 1.17) poly(oligo(2‐ethyl‐2‐oxazine) methacrylate) P(OEtOzMA)n brushes with tunable lower critical solution temperature (LCST) behavior and negligible cell toxicity is prepared. Upon deprotection, the phosphonic acid end‐group of the P(OEtOzMA)n brush enables the successful grafting‐onto iron oxide nanoparticles (IONPs). Colloidal stability of the particle suspension in combination with suitable magnetic resonance imaging (MRI) relaxivities demonstrates the potential of these particles for future applications as negative MRI contrast agents.\ud \ud

A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.

Protecting the heart after an acute coronary syndrome is a key therapeutic goal to support cardiac recovery and prevent progression to heart failure. A potential strategy is to target cardiac glucose metabolism at the early stages after ischemia when glycolysis is critical for myocyte survival. Building on our discovery that high-density lipoprotein (HDL) modulates skeletal muscle glucose metabolism, we now demonstrate that a single dose of reconstituted HDL (rHDL) delivered after myocardial ischemia increases cardiac glucose uptake, reduces infarct size, and improves cardiac remodeling in association with enhanced functional recovery in mice. These findings applied equally to metabolically normal and insulin-resistant mice. We further establish direct effects of HDL on cardiomyocyte glucose uptake, glycolysis, and glucose oxidation via the Akt signaling pathway within 15 min of reperfusion. These data support the use of infusible HDL preparations for management of acute coronary syndromes in the setting of primary percutaneous interventions.

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.

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.

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 scFv(anti-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 (64)Cu. 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.

Rationale: 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. Objective: We developed a novel targeted fibrinolytic drug that is directed against activated platelets. Methods and Results: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFv SCE5 ), which targets the activated form of the platelet–integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFv SCE5 –scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline ( P SCE5 –scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFv SCE5 –scuPA as compared with the same dose of a mutated, nontargeting scFv–scuPA or vehicle. The ability of scFv SCE5 –scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. Conclusions: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.

Introduction 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. Methods 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. Results 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 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 5minutes after injection (p Conclusions Our results show that the novel antibody radiotracer 18 F-scFv anti-LIBS is useful for the sensitive detection of activated platelets and thrombosis. Advances in knowledge and implications for patient care 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.

Background Thrombolytic therapy for acute thrombosis is limited by life‐threatening side effects such as major bleeding and neurotoxicity. New treatment options with enhanced fibrinolytic potential are therefore required. Here, we report the development of a new thrombolytic molecule that exploits key features of thrombosis. We designed a recombinant microplasminogen modified to be activated by the prothrombotic serine‐protease thrombin (HtPlg), fused to an activation‐specific anti–glycoprotein IIb/ III a single‐chain antibody ( SCE 5), thereby hijacking the coagulation system to initiate thrombolysis. Methods and Results The resulting fusion protein named SCE 5‐HtPlg shows in vitro targeting towards the highly abundant activated form of the fibrinogen receptor glycoprotein IIb/ III a expressed on activated human platelets. Following thrombin formation, SCE 5‐HtPlg is activated to contain active microplasmin. We evaluate the effectiveness of our targeted thrombolytic construct in two models of thromboembolic disease. Administration of SCE 5‐HtPlg (4 μg/g body weight) resulted in effective thrombolysis 20 minutes after injection in a ferric chloride–induced model of mesenteric thrombosis (48±3% versus 92±5% for saline control, P P SCE 5‐HtPlg did not prolong bleeding time compared with saline ( P =0.99). Conclusions Our novel fusion molecule is a potent and effective treatment for thrombosis that enables in vivo thrombolysis without bleeding time prolongation. The activation of this construct by thrombin generated within the clot itself rather than by a plasminogen activator, which needs to be delivered systemically, provides a novel targeted approach to improve thrombolysis.

The prostate-specific membrane antigen (PSMA) is abundantly expressed on prostate cancer epithelial cells and its expression correlates with tumor progression. Therefore, a specific immunotherapy against this antigen may be a novel therapeutic option for the management of prostate cancer. We generated an anti-PSMA single-chain antibody fragment (scFv), called D7, by phage display from the monoclonal antibody 3/F11. By C-terminal ligation of the toxic domain of Pseudomonas Exotoxin A (PE40) to the genes of D7, the immunotoxin D7-PE40 was generated. D7 and D7-PE40 specifically bound to PSMA transfectants and to the PSMA expressing prostate cancer cell line C4-2. In addition, D7-PE40 showed a high serum stability and induced a 50% reduction of viability (IC50) in C4-2 cells at a concentration of 140 pM. In vivo, D7-PE40 was well tolerated in SCID mice up to a single dose of 20 microg, whereas higher doses induced severe hepatotoxicity with deaths of the animals. Immunotoxin treatment of mice bearing C4-2 tumor xenografts caused a significant inhibition of tumor growth, whereas mice with PSMA-negative DU 145 tumors remained unaffected. Owing to its high and specific cytotoxicity and its capability to inhibit prostate tumor growth in vivo the immunotoxin D7-PE40 represents a promising candidate for the immunotherapy of prostate cancer.

BACKGROUND The prostate specific membrane antigen (PSMA) represents an attractive antigen for antibody-based diagnostic and therapeutic intervention in prostate cancer, since it is highly restricted to the prostate and overexpressed in all tumor stages. The present work describes the in vitro characterization of the three anti-PSMA monoclonal antibodies (mAbs) 3/A12, 3/E7, and 3/F11 in comparison to the mAb J591. METHODS The mAbs were tested for saturation and competitive binding on C4-2 prostate cancer cells by flow cytometry. Immunohistochemical staining was conducted on frozen prostate normal and cancer tissues as well as on lymph node metastases. Similarly, potential crossreactivities were tested on a broad panel of human normal tissues. RESULTS The anti-PSMA mAbs showed a strong binding to C4-2 cells with mean half-maximal saturation concentrations of about 14 nM for 3/A12, 17 nM for 3/E7, 9 nM for 3/F11, and 16 nM for J591. Competitive binding studies revealed that our three mAbs bind to different extracellular PSMA epitopes. The mAbs showed comparable staining of epithelial cells for all tested normal and tumorous prostate tissues. Extraprostatic staining was observed on secretory cells of the salivary glands and on the brush border of the duodenal columnar epithelium. J591 additionally showed positive staining of the normal breast epithelium. CONCLUSIONS Due to their specific binding characteristics, the anti-PSMA mAbs 3/A12, 3/E7, and 3/F11 show great promise for diagnostic and therapeutic applications in prostate cancer. Prostate 70: 562–569, 2010. © 2009 Wiley-Liss, Inc.

BACKGROUND Expression of the prostate specific membrane antigen (PSMA) is highly restricted to prostate epithelial cells. Therefore, toxin-based immunotherapy against this antigen may represent an alternative therapeutic option for prostate cancer. For these purposes, the effects of the recombinant anti-PSMA immunotoxin A5-PE40 on prostate tumor growth were investigated in vitro and in vivo. METHODS The in vitro binding and cytotoxicity of A5-PE40 were tested on the PSMA-expressing prostate cancer cell line C4-2 and on the PSMA-negative cell line DU145 by flow cytometry and WST assays. The binding of the immunotoxin to SCID mouse xenografts and to various mouse organs was examined by Western blot analysis. In vivo, the antitumor activity of the immunotoxin was tested by injecting A5-PE40 in mice bearing C4-2 or DU145 xenografts. RESULTS In vitro, a specific binding of A5-PE40 to C4-2 cells could be shown with a concentration-dependent cytotoxicity (IC50 value = 220 pM). In the next step, a specific binding of the immunotoxin to C4-2 xenografts could be demonstrated. In contrast, no binding on mouse organs expressing high homologous mouse PSMA was found. The treatment of mice with C4-2 tumors caused a significant inhibition of tumor growth in vivo, whereas DU145 xenografts remained totally unaffected. CONCLUSIONS A5-PE40 represents a recombinant anti-PSMA immunotoxin with potent antitumor activity in mice bearing human prostate cancer xenograft tumors. Therefore, A5-PE40 could be a promising candidate for therapeutic applications in patients with prostate cancer. Prostate 68: 129–138, 2008. © 2007 Wiley-Liss, Inc.

Aims: High-density lipoprotein (HDL) is known to increase skeletal muscle glucose uptake, but whether this action extends to the myocardium and has relevance in the setting of ischemia is unknown. This study aimed to determine the effect of HDL on cardiac glucose metabolism both in vitro and in vivo, including during ischemia/reperfusion injury. Methods and Results: HDL treatment (50μg/mL) increased glucose uptake (147±10%), glycolysis (154±11%) and glucose oxidation (130±6%) in neonatal ventricular cardiomyocytes compared to saline control. HDL rapidly activated the Akt pathway and an Akt inhibitor (Akti 1/2) abolished HDL-mediated glucose uptake, suggesting an Akt-dependent mechanism. To determine the upstream receptor mechanism HDL binding to sphingosine-1-phosphate (S1P) receptors was blocked with either 1uM VPC23019 (S1P1&3 antagonist) or 1uM CAY10444 (S1P3 antagonist). Both similarly inhibited HDL-mediated glucose uptake. S1P is a known activator of the Akt pathway, thus our data suggests the S1P component of HDL increases glucose uptake via S1P3 activation of the Akt pathway. The effect of a single intravenous bolus of reconstituted HDL (CSL-111; 80mg/kg) in chow diet (CD) and high fat diet (HFD) insulin resistant C57Bl6 mice was investigated during an oral glucose tolerance test (OGTT). CSL-111 treatment increased cardiac muscle glucose clearance in both CD (168±16%) and HFD (138±11%) mice compared to saline infusion during the OGTT. To assess whether CSL-111 could increase cardiac glucose uptake in an ischemic setting, a single bolus of CSL-111 (80mg/kg) was given at the beginning of reperfusion in mice following left anterior descending artery (LAD) ligation. CSL-111 trended to increase cardiac glucose uptake ([18F]Fluorodeoxyglucose tracer and PET-CT imaging) by 66% (chow) and 81% (HFD) (treatment effect p=0.07) 1 hour post-reperfusion following LAD surgery. Furthermore, CSL-111 improved long term cardiac functional recovery. Conclusion: These data show that HDL increases glucose uptake and utilisation in the heart suggesting a role for HDL therapies in preserving functional myocardium subsequent to acute coronary ischemic syndromes.

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 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.

We report the engineering of poly(ethylene glycol) (PEG) hydrogel particles using a mesoporous silica (MS) templating method via tuning the PEG molecular weight, particle size, and the presence or absence of the template and investigate the cell association and biodistribution of these particles. An ex vivo assay based on human whole blood that is more sensitive and relevant than traditional cell-line based assays for predicting in vivo circulation behavior is introduced. The association of MS@PEG particles (template present) with granulocytes and monocytes is higher compared with PEG particles (template absent). Increasing the PEG molecular weight (from 10 to 40 kDa) or decreasing the PEG particle size (from 1400 to 150 nm) reduces phagocytic blood cell association of the PEG particles. Mice biodistribution studies show that the PEG particles exhibit extended circulation times (>12 h) compared with the MS@PEG particles and that the retention of smaller PEG particles (150 nm) in blood, when compared with larger PEG particles (>400 nm), is increased at least 4-fold at 12 h after injection. Our findings highlight the influence of unique aspects of polymer hydrogel particles on biological interactions. The reported PEG hydrogel particles represent a new class of polymer carriers with potential biomedical applications.

Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.

Therapeutic nanoparticles hold clinical promise for cancer treatment by avoiding limitations of conventional pharmaceuticals. Herein, a facile and rapid method is introduced to assemble poly(ethylene glycol) (PEG)-modified Pt prodrug nanocomplexes through metal-polyphenol complexation and combined with emulsification, which results in ≈100 nm diameter nanoparticles (PtP NPs) that exhibit high drug loading (0.15 fg Pt per nanoparticle) and low fouling properties. The PtP NPs are characterized for potential use as cancer therapeutics. Mass cytometry is used to quantify uptake of the nanoparticles and the drug concentration in individual cells in vitro. The PtP NPs have long circulation times, with an elimination half-life of ≈18 h in healthy mice. The in vivo antitumor activity of the PtP NPs is systematically investigated in a human prostate cancer xenograft mouse model. Mice treated with the PtP NPs demonstrate four times better inhibition of tumor growth than either free prodrug or cisplatin. This study presents a promising strategy to prepare therapeutic nanoparticles for biomedical applications.

The goal of the present study was to test the (177)Lu-labelled anti-PSMA monoclonal antibody 3/F11 ((177)Lu-DOTA-3/F11) as a new radioimmunotherapeutic agent in a prostate cancer SCID mouse xenograft model.The mAb 3/F11 was (177)Lu labelled using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as chelating agent. DOTA-3/F11 was tested for cell binding and serum immunoreactivity by flow cytometry. The biodistribution and the therapeutic efficacy of (177)Lu-DOTA-3/F11 in mice bearing PSMA-positive C4-2 prostate cancer xenografts were evaluated.3/F11 and DOTA-3/F11 showed high and specific cell binding and similar serum half-lives of approximately seven days. Biodistribution studies revealed an increasing tumour uptake of (177)Lu DOTA-3/F11 over time with maximum tumour-to-muscle and tumour-to-blood ratios after 72 h. A single dose of 1 MBq (177)Lu-DOTA-3/F11 inhibited tumour growth and prolonged survival.This study indicated that (177)Lu-DOTA-3/F11 may be a suitable radioimmunotherapeutic agent for the treatment of prostate cancer.

BACKGROUND The prostate specific membrane antigen (PSMA) is expressed by virtually all prostate cancers and represents an ideal target for diagnostic and therapeutic strategies. This article compares the in vivo behavior and tumor uptake of three different radiolabeled anti-PSMA monoclonal antibodies (mAbs) and corresponding F(ab)2 and Fab fragments thereof. METHODS The mAbs 3/A12, 3/F11, and 3/E7 and fragments of 3/A12 were conjugated with the chelating agent DOTA and radiolabeled with 64Cu. For the microPET imaging studies, SCID mice bearing PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts were used. Each animal received 20–30 µg radiolabeled mAb or fragment corresponding to an activity of 8–14 MBq. Imaging was performed 3, 24, and 48 hr post-injection. After the last scan, mice were sacrificed and tracer in vivo biodistribution was measured by gamma-counting. RESULTS Static microPET images of mice with PSMA-positive tumors revealed a high uptake of the mAbs in the C4-2 tumors at 24 and 48 hr after tracer injection and only a minimal distribution in the DU 145 tumors and other organs. In contrast, the F(ab)2 and Fab fragments of 3/A12 were detected at a high extend in the kidney but not in the C4-2 tumors. These results were confirmed by gamma counting of dissected organs after the final imaging. CONCLUSIONS Due to the high and specific uptake of the 64Cu-labeled mAbs in PSMA-positive tumors, these antibodies represent excellent tools for prostate cancer imaging. Prostate 70: 1413–1421, 2010. © 2010 Wiley-Liss, Inc.

The prostate specific membrane antigen (PSMA) represents an attractive antigen for antibody-based diagnostic and therapeutic intervention in prostate cancer, since it is highly restricted to the prostate and overexpressed in all tumor stages. The present work describes the in vitro characterization of the three anti-PSMA monoclonal antibodies (mAbs) 3/A12, 3/E7, and 3/F11 in comparison to the mAb J591.The mAbs were tested for saturation and competitive binding on C4-2 prostate cancer cells by flow cytometry. Immunohistochemical staining was conducted on frozen prostate normal and cancer tissues as well as on lymph node metastases. Similarly, potential crossreactivities were tested on a broad panel of human normal tissues.The anti-PSMA mAbs showed a strong binding to C4-2 cells with mean half-maximal saturation concentrations of about 14 nM for 3/A12, 17 nM for 3/E7, 9 nM for 3/F11, and 16 nM for J591. Competitive binding studies revealed that our three mAbs bind to different extracellular PSMA epitopes. The mAbs showed comparable staining of epithelial cells for all tested normal and tumorous prostate tissues. Extraprostatic staining was observed on secretory cells of the salivary glands and on the brush border of the duodenal columnar epithelium. J591 additionally showed positive staining of the normal breast epithelium.Due to their specific binding characteristics, the anti-PSMA mAbs 3/A12, 3/E7, and 3/F11 show great promise for diagnostic and therapeutic applications in prostate cancer.

Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein, is highly expressed by virtually all prostate cancers and is currently the focus of several diagnostic and therapeutic strategies. We have previously reported on the generation of several monoclonal antibodies (mAb) and antibody fragments that recognize and bind with high affinity to the extracellular domain of cell-adherent PSMA. This article reports the in vivo behavior and tumor uptake of the radiolabeled anti-PSMA mAb 3/A12 and its potential as a tracer for PET. Methods: The mAb 3/A12 was conjugated with the chelating agent 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) and radiolabeled with 64Cu. Severe combined immunodeficient mice bearing PSMA-positive C4-2 prostate carcinoma xenografts were used for small-animal PET imaging. Mice with PSMA-negative DU 145 tumors served as controls. For PET studies, each animal received 20–30 μg of radiolabeled mAb corresponding to an activity of 7.6–11.5 MBq. Imaging was performed 3, 24, and 48 h after injection. After the last scan, the mice were sacrificed and tracer in vivo biodistribution was measured by γ-counting. Results: Binding of the mAb 3/A12 on PSMA-expressing C4-2 cells was only minimally influenced by DOTA conjugation. The labeling efficiency using 64Cu and DOTA-3/A12 was 95.3% ± 0.3%. The specific activity after 64Cu labeling was between 327 and 567 MBq/mg. After tracer injection, static small-animal PET images of mice with PSMA-positive tumors revealed a tumor-to-background ratio of 3.3 ± 1.3 at 3 h, 7.8 ± 1.4 at 24 h, and 9.6 ± 2.7 at 48 h. In contrast, no significant tracer uptake occurred in the PSMA-negative DU 145 tumors. These results were confirmed by direct counting of tissues after the final imaging. Conclusion: Because of the high and specific uptake of 64Cu-labeled mAb 3/A12 in PSMA-positive tumors, this ligand represents an excellent candidate for prostate cancer imaging and potentially for radioimmunotherapy.