Bivalent engagement of endothelial surface antigens is critical to prolonged surface targeting and protein delivery in vivo.

Targeted drug delivery to the endothelium has the potential to generate localized therapeutic effects at the blood-tissue interface. For some therapeutic cargoes, it is essential to maintain contact with the bloodstream to exert protective effects. The pharmacokinetics (PK) of endothelial surface-targeted affinity ligands and biotherapeutic cargo remain a largely unexplored area, despite obvious translational implications for this strategy. To bridge this gap, we site-specifically radiolabeled mono- (scFv) and bivalent (mAb) affinity ligands specific for the endothelial cell adhesion molecules, PECAM-1 (CD31) and ICAM-1 (CD54). Radiotracing revealed similar lung biodistribution at 30 minutes post-injection (79.3% ± 4.2% vs 80.4% ± 10.6% ID/g for αICAM and 58.9% ± 3.6% ID/g vs. 47.7% ± 5.8% ID/g for αPECAM mAb vs. scFv), but marked differences in organ residence time, with antibodies demonstrating an order of magnitude greater area under the lung concentration vs. time curve (AUCinf 1698 ± 352 vs. 53.3 ± 7.9 ID/g*hrs for αICAM and 1023 ± 507 vs. 114 ± 37 ID/g*hrs for αPECAM mAb vs scFv). A physiologically based pharmacokinetic model, fit to and validated using these data, indicated contributions from both superior binding characteristics and prolonged circulation time supporting multiple binding-detachment cycles. We tested the ability of each affinity ligand to deliver a prototypical surface cargo, thrombomodulin (TM), using one-to-one protein conjugates. Bivalent mAb-TM was superior to monovalent scFv-TM in both pulmonary targeting and lung residence time (AUCinf 141 ± 3.2 vs 12.4 ± 4.2 ID/g*hrs for ICAM and 188 ± 90 vs 34.7 ± 19.9 ID/g*hrs for PECAM), despite having similar blood PK, indicating that binding strength is more important parameter than the kinetics of binding. To maximize bivalent target engagement, we synthesized an oriented, end-to-end anti-ICAM mAb-TM conjugate and found that this therapeutic had the best lung residence time (AUCinf 253 ± 18 ID/g*hrs) of all TM modalities. These observations have implications not only for the delivery of TM, but also potentially all therapeutics targeted to the endothelial surface.

Vascular endothelial effects of collaborative binding to platelet/endothelial cell adhesion molecule-1 (PECAM-1).

Targeting drugs to endothelial cells has shown the ability to improve outcomes in animal models of inflammatory, ischemic and thrombotic diseases. Previous studies have revealed that certain pairs of ligands (antibodies and antibody fragments) specific for adjacent, but distinct, epitopes on PECAM-1 enhance each other's binding, a phenomenon dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL. This discovery has been leveraged to enable simultaneous delivery of multiple therapeutics to the vascular endothelium. Given the known role of PECAM-1 in promoting endothelial quiescence and cell junction integrity, we sought here to determine if CEPAL might induce unintended vascular effects. Using a combination of in vitro and in vivo techniques and employing human and mouse endothelial cells under physiologic and pathologic conditions, we found only modest or non-significant effects in response to antibodies to PECAM-1, whether given solo or in pairs. In contrast, these methods detected significant elevation of endothelial permeability, pro-inflammatory vascular activation, and systemic cytokine release following antibody binding to the related endothelial junction protein, VE-Cadherin. These studies support the notion that PECAM-1-targeted CEPAL provides relatively well-tolerated endothelial drug delivery. Additionally, the analysis herein creates a template to evaluate potential toxicities of vascular-targeted nanoparticles and protein therapeutics.

Increased protein glycation in cerebrospinal fluid of Alzheimer's disease.

Accumulation of advanced glycation end products occurs in the brain with ageing and was proposed to be involved in pathogenesis of Alzheimer's disease. We studied changes in the level of an early glycation product, an Amadori product, in cerebrospinal fluid (CSF) in ageing and in late-onset Alzheimer's disease. The work was carried out on 99 consecutive patients. The concentration of Amadori product in CSF correlated with CSF glucose concentration but was not changed with age (n = 70). In contrast, level of CSF Amadori product was 1.7-fold higher in Alzheimer's disease patients (n = 29) as compared with non-demented age-matched control group (n = 20; P < 0.0005), although CSF glucose concentration was similar in both groups (4.1 +/- 1.3 vs. 3.8 +/- 0.6 mmol/liter, resp.). An increased accumulation of Amadori products was found in all major proteins of CSF of Alzheimer's disease including albumin, apolipoprotein E and transthyretin. We propose that the increased early glycation of CSF proteins in the Alzheimer's patients may stimulate the formation and the consequent deposition of advanced glycation end products as well as oxidative stress in the brain.

Heparin specifically inhibits binding of apolipoprotein E to amyloid beta-peptide.

Apolipoprotein E (apoE) binds to non-fibrillar amyloid beta-peptide with high affinity. We find here that heparin specifically inhibits apoE-amyloid beta-peptide (1-40) interaction. Low molecular weight heparins reduce the affinity of this interaction 3-fold as it was estimated by surface plasmon resonance. The binding is not affected by high salt concentration, which prevents heparin-induced changes of apoE conformation. We propose that rigid protein conformation, induced by high affinity heparin binding to apoE, is unfavorable for its interaction to amyloid beta-peptide. Using thioflavin T assay, we find that heparin promotes fibrillogenesis of amyloid beta-peptide whereas apoE abolishes this effect. The data suggests that the relationship between apoE and glycosaminoglycans may be important for amyloid beta-peptide fibril formation.

Kinetics of apolipoprotein E isoforms-binding to the major glycosaminoglycans of the extracellular matrix.

Apolipoprotein E (apoE), a key lipid transport protein, displays a heparin-binding property that is critical in several apoE functions. The kinetics of the interaction between apoE isoforms and glycosaminoglycans (GAGs) were studied using surface plasmon resonance. The dissociation constant of equilibrium K(D) for apoE3-heparin interaction was estimated to be 12 nM for apoE3 and three common apoE isoforms revealed similar affinities for heparin. ApoE binds to GAGs in the following order: heparin>heparan sulfate>dermatan sulfate>chondroitin sulfate. The affinity parameter of the binding of low molecular weight heparins to apoE is correlated with the chain length. The effective number Z of electrostatic interactions between plasma apoE3 and heparin was assessed to be three. Metal chelators were able to diminish apoE-binding to heparin, suggesting some stabilizing effect of metal ions while reconstitution with lipids did not affect binding affinities for heparin, suggesting that the N-terminal heparin-binding site is responsible for apoE-containing lipoprotein interactions with heparin.

Capillary electrophoretic analysis of recombinant human apolipoprotein E. Calibration mode of a protein reference material.

Actual lack of standardization of serum apolipoprotein (apo) E measurements prevents intensive studies on the significance of apoE concentration in clinical chemistry. For this purpose the use of standards calibrated against a common reference material is essential in order to obtain reliable results. The assignment of a certified value to this reference material involves the use of an accurate and non immunological technique. We demonstrate here that capillary electrophoresis in sodium dodecyl sulfate containing gel (SDS-CGE) can be a method of choice. ApoE quantification was performed according to the peak area and a standard solution of purified apoAI. We obtained very close results to that obtained by HPLC of phenylalanine apoE content measurement i.e., 81.6 mg/l versus 84.6 mg/l and concluded that the SDS-CGE analysis is an accurate and reliable method for the certification of an apoE reference material to be used in serum apoE concentration measurements. The SDS-CGE combines advantages of SDS-polyacrylamide gel electrophoresis (high resolving power, rapidity and tolerance of complex sample) with that of quantitative HPLC analysis (accuracy, precision, linearity and speed).

Glycation of apolipoprotein E impairs its binding to heparin: identification of the major glycation site.

The increased glycation of plasma apolipoproteins represents a possible major factor for lipid disturbances and accelerated atherogenesis in diabetic patients. The glycation of apolipoprotein E (apoE), a key lipid-transport protein in plasma, was studied both in vivo and in vitro. ApoE was shown to be glycated in plasma very low density lipoproteins of both normal subjects and hyperglycemic, diabetic patients. However, diabetic patients with hyperglycemia showed a 2-3-fold increased level of apoE glycation. ApoE from diabetic plasma showed decreased binding to heparin compared to normal plasma apoE. The rate of Amadori product formation in apoE in vitro was similar to that for albumin and apolipoproteins A-I and A-II. The glycation of apoE in vitro significantly decreased its ability to bind to heparin, a critical process in the sequestration and uptake of apoE-containing lipoproteins by cells. Diethylenetriaminepentaacetic acid, a transition metal chelator, had no effect on the loss of apoE heparin-binding activity, suggesting that glycation rather than glycoxidation is responsible for this effect. In contrast, glycation had no effect on the interaction of apoE with amyloid beta-peptide. ApoE glycation was demonstrated to be isoform-specific. ApoE(2) showed a higher glycation rate and the following order was observed: apoE(2)>apoE(4)>apoE(3). The major glycated site of apoE was found to be Lys-75. These findings suggest that apoE is glycated in an isoform-specific manner and that the glycation, in turn, significantly decreases apoE heparin-binding activity. We propose that apoE glycation impairs lipoprotein-cell interactions, which are mediated via heparan sulfate proteoglycans and may result in the enhancement of lipid abnormalities in hyperglycemic, diabetic patients.

Interaction between human amphipathic apolipoproteins and amyloid beta-peptide: surface plasmon resonance studies.

Several apolipoproteins including apoE and apoA-I are known to be associated with amyloid beta-peptide, a major component of senile plaques in Alzheimer's disease. In the present study the interaction between three human amphipathic apolipoproteins apoE3, apoA-I and apoA-II and immobilized amyloid beta-peptide (1-40) was quantified by plasmon resonance. The interactions were saturable and reversible. The results demonstrated a high affinity of the binding of amphipathic apolipoproteins to amyloid beta-peptide. On the other hand, only a small population of synthetic amyloid beta-peptide participated in the interaction. The apparent equilibrium dissociation constants K(D) were 10 nM for apoE3, 25 nM for apoA-I and 80 nM for apoA-II under physiological conditions. The affinity of the apoE3-amyloid beta-peptide binding was not affected by pH in the range 6.0-8.0 but was significantly increased by high salt concentration. ApoA-I mainly followed similar patterns. A major participation of hydrophobic forces in the binding of apoE3 and apoA-I to amyloid beta-peptide was suggested.

[The immunochemical detection of apoprotein dissociation from particles of very low-density lipoproteins in human blood plasma]. / Immunokhimicheskoe obnaruzhenie otdeleniia apobelkov ot chastits lipoproteinov ochen' nizkoi plotnosti plazmy krovi cheloveka.

The dissociation of very-low-density lipoprotein (VLDL) apoproteins was studied using immunochemical approaches. The analysis of monospecific antibody binding to apo E, C-II and C-III on VLDL surface showed low apoprotein accessibility for the antibodies while the accessibility of apo C-II and C-III in solution was complete. Lipoprotein preparation dilution resulted in increasing of apo E and C-II accessibility. It was suggested that apoprotein dissociation led to apoprotein cluster dissolving on VLDL surface and higher antigen determinant accessibility. The findings confirmed previous theoretical analysis of apoprotein dissociation.

Quaternary structure of apolipoprotein E in solution: fluorimetric, chromatographic and immunochemical studies.

Self-association and stability in solution of apolipoprotein E (apoE), isolated from human plasma very low density lipoproteins, were studied in the nanomolar concentration range. Equilibrium denaturation of fluorescein-labelled apoE (induced by guanidine hydrochloride) was studied by measurement of fluorescence anisotropy, total fluorescence emission intensity, the shift in wavelength of maximal fluorescence emission, and gel-chromatographic behaviour. The protein denaturation was reversible, displayed biphasic behaviour, and was dependent on the apoE concentration. As measured by fluorescence anisotropy, the kinetics of apoE denaturation in the presence of 6M denaturant were heterogeneous, and the contribution of the long-lived component increased with the apoprotein concentration. The results are in agreement with the following scheme: Oligomer (in aged preparations) in equilibrium with tetramer in equilibrium with native or partially denatured monomer in equilibrium with fully denatured monomer. It is suggested that self-association of individual apoE molecules in solution is due to their lipid-binding domains, and leads to additional stabilization of apoprotein structure. Monoclonal antibody 3D12F11 of the IgG1 subclass bound with high affinity to apoE (Kd = 3.5 +/- 0.5 nM), and had no effect on apoprotein binding to heparin-Sepharose or the apoprotein-induced destabilization of liposomes formed from dipalmitoyl-phosphatidylcholine. This indicates that the epitope to the antibody is localized outside the heparin- and lipid-binding sites of the apoprotein molecule.

[Preparation and characterization of monoclonal antibodies to human apolipoprotein E]. / Poluchenie i kharakteristika monoklonal'nykh antitel protiv apolipoproteina E cheloveka.

Monoclonal antibodies to human apolipoprotein E (apoE) were prepared and characterized. Antibodies of 3D12F11 clone were shown to be specific to apoE and belong to IgG1 subclass. Dissociation constant of antigen-antibody complex in solution was determined as 3.5 +/- 0.5 nM. The antibodies interacted neither heparin- nor lipid-binding sites of human apoE molecule. The obtained antibodies may be useful for metabolic and structural investigations of apoE as well as for clinical studies.

[Supermolecular organization of apolipoprotein E in solution]. / Nadmolekuliarnaia organizatsiia apolipoproteina E v rastvore.

The aggregation behaviour and stability in solution of apolipoprotein E (apoE) isolated from human blood plasma very low density lipoproteins were investigated. The equilibrium denaturation of fluorescein-labeled apoE by guanidine-hydrochloride determined by anisotropy and overall intensity of fluorescence, shift of the emission spectrum maximum and gel-chromatographic behaviour was characterized by reversibility, biphasity, apoE concentration dependence and the existence of native structure of the apoE monomer. The contribution of the long-living component to the kinetic dependence of fluorescence anisotropy in the presence of the 6 M denaturant increased with an increase in apoE concentration. The data obtained fit into the following scheme: oligomer (upon aging of the preparation) in equilibrium tetramer in equilibrium native monomer in equilibrium denaturated monomer. The presence in the tetrameric structure of apoE of two domains is postulated; one of those is formed by lipid-binding fragments during aggregation of individual molecules of apoE. Monoclonal antibody 3D12F11 (subclass IgG1) showed a high affinity for the apoE (Kd = 3.5 +/- 0.5 nM) without any effect on the apoprotein binding to heparin-Sepharose and apoE-induced destruction of dipalmitoylphosphatidylcholine liposomes. It is concluded that the 3D12F11 epitope is localized outside heparin- and lipid-binding sites of the apoprotein molecule.

[Heparin-sepharose affinity chromatography and reduction conditions as a method of a selective process for separate isoforms of apolipoprotein A]. / Affinnaia khromatografiia na geparin-sefaroze v redutsiruiushchikh usloviiakh kak sposob izbiratel'nogo obogashcheniia po otdel'nym izoformam apolipoproteina E.

Gel permeation chromatography of VLDL apoproteins on Sepharose CL-6B in denaturing conditions and affinity chromatography on heparin-sepharose 4B in the presence of reducing agent dithiothreitol were used for preparative isolation of apolipoprotein E of high purity from human plasma VLDL. Sequential elution of apolipoprotein E from affinity column using increasing ionic strength solutions (0.4 M NaCl and 1.0 M NaCl) permitted to obtain "high affinity" apo E preparation with increased relative apoprotein isoform content with the highest positive charge.

[Dynamic behavior of apoproteins of human plasma very low density lipoproteins and lipolysis regulation]. / Dinamicheskoe povedenie apobelkov lipoproteidov ochen' nizkoi plotnosti plazmy krovi cheloveka i reguliatsiia protsessa lipoliza.

Using the dynamic fluorescence quenching method, it was shown that very low density (VLDL) apoproteins (apo B, E and C) tryptophanyls exhibit a lower accessibility towards water-soluble quenchers as compared to apo B LDL chromophores. The efficiency of proteolytic degradation by trypsin of VLDL-associated apo E and apo C was much lower than that of apo B. These results may be due to the cluster arrangement of amphipatic apo E and apo C on the VLDL surface and/or to their partial shielding by apo B. Treatment of VLDL particles with sub-lytic concentrations of the detergent, Tween-20, did not change the relaxation characteristics of amphipatic apoprotein tryptophanyl microenvironment, but resulted in a reversible structural transition registered by a "red" shift of the emission spectrum maximum as well as by change of the iodine quenching pattern. The detergent-induced increase of the VLDL tryptophanyl accessibility to acrylamide and the decrease of the quenching constant at the partial and complete particle solubilization were related to a change of the apo B molecular package. Treatment of VLDL with Tween-20 or cow milk lipoprotein lipase resulted in the appearance of tryptophanyl population that was not involved in the resonance energy transfer to the lipid phase-localized fluorescent probe pyrene, which is indicative of the protein dissociation. Treatment of VLDL particles with sub-lytic concentrations of Tween-20 revealed a lower (compared to apo C) relative affinity of apo E for the VLDL lipid surface. Inhibition of the lipoprotein lipase activity by apoprotein C-III was found to be non-competitive. It was concluded that lipolysis is a self-regulatory process which involves changes in the effector apoprotein concentration on the surface of triglyceride-rich particles.

Topo-dynamic characteristics of human plasma VLDL apolipoproteins and efficiency of triacylglycerol hydrolysis by lipoprotein lipase.

A lower accessibility to water-soluble quenchers of tryptophanyls of VLDL apolipoproteins B, E, C as compared to LDL apoB chromophores has been detected by a fluorescence quenching technique. The dynamic behaviour of the tryptophanyls of VLDL amphipathic apolipoproteins E and C did not change in the presence of a detergent, Tween-20, at sub-lytic concentrations. However, a reversible structural transition registered by the 'red' shift of the emission spectrum maximum and the changes in the quenching pattern by I- occurred under these conditions. The increase in the VLDL tryptophanyl accessibility to acrylamide and the decrease in the quenching constant were observed at partial and complete solubilization of the VLDL particles by the detergent. Dissociation of apolipoproteins from VLDL occurred after their treatment with Tween-20 or lipoprotein lipase isolated from bovine milk, and the tryptophanyl population not participating in fluorescence energy transfer on lipid phase-localized fluorescent probe pyrene appeared. In the presence of Tween-20, the relative affinity of apoE for the lipid matrix of VLDL was lower than that of apoC. Besides, the uncompetitive mode of inhibition of the LPL activity by apoC-III has been demonstrated. It is suggested that: (1) the amphipathic apolipoproteins E and C are organized as clusters on the VLDL surface and/or partially shielded by apolipoprotein B: (2) self-regulation of lypolysis may exist involving detergent-like reaction product accumulation and changes in relative apolipoprotein contents.

VLDL substrate properties and efficiency of their metabolic transformation by LPL.

A study was made of the regulation of the triglyceride hydrolysis catalysed by LPL from bovine milk, by the apoproteins from human plasma VLDL. Both isolated apolipoproteins, and those found on the surface of plasma VLDL particles, were investigated. A concentration-dependent activating action of apo C-II on the hydrolysis of emulsified triolein, and uncompetitive inhibition of VLDL triglyceride hydrolysis by apo C-III were found. It is suggested that VLDL lipolysis might be controlled in vivo through the variation of the relative surface content of these enzymatic activity modulators.