Wim K. Bleeker

Effect of Target Dynamics on Pharmacokinetics of a Novel Therapeutic Antibody against the Epidermal Growth Factor Receptor: Implications for the Mechanisms of Action

The epidermal growth factor receptor (EGFR) is overexpressed on many solid tumors and represents an attractive target for antibody therapy. Here, we describe the effect of receptor-mediated antibody internalization on the pharmacokinetics and dose-effect relationship of a therapeutic monoclonal antibody (mAb) against EGFR (2F8). This mAb was previously found therapeutically active in mouse tumor models by two dose-dependent mechanisms of action: blockade of ligand binding and induction of antibody-dependent cell-mediated cytotoxicity. In vitro studies showed 2F8 to be rapidly internalized by EGFR-overexpressing cells. In vivo, accelerated 2F8 clearance was observed in cynomolgus monkeys at low doses but not at high doses. This enhanced clearance seemed to be receptor dependent and was included in a pharmacokinetic model designed to explain its nonlinearity. Receptor-mediated clearance was also found to affect in situ antibody concentrations in tumor tissue. Ex vivo analyses of xenograft tumors of 2F8-treated nude mice revealed that relatively high antibody plasma concentrations were required for maximum EGFR saturation in high-EGFR-expressing human A431 tumors, in contrast to lower-EGFR-expressing human xenograft tumors. In summary, receptor-mediated antibody internalization and degradation provides a saturable route of clearance that significantly affects pharmacokinetics, particularly at low antibody doses. EGFR saturation in normal tissues does not predict saturation in tumor tissue as local antibody concentrations in EGFR-overexpressing tumors may be more rapidly reduced by antibody internalization. Consequently, antibody saturation of the receptor may be affected, thereby affecting the local mechanism of action.

Pacemaker cell types in the rabbit sinus node: A correlative ultrastructural and electrophysiological study

In isolated preparations of the rabbit sinus node, we have investigated the fine structure of the tissue at many sites which had been electro-physiologically identified by means of microelectrode recordings. For each of these sites we quantified the myofilament density of the cells, since this appeared to be a useful parameter for characterizing cell types in the sinus node region; and because myofilaments were present both in isolated form and organized in myofibrils, the degree of organization was also measured in a semi-quantitative fashion. The electrical activity of cells at a given site was characterized by the activation moment relative to the cardiac cycle and furthermore by the rate of diastolic depolarization and the maximum rate of rise of the action potential. From the centre of the node toward the periphery a very gradual increase in myofilament density was observed in all directions. It was found that the rate of diastolic depolarization, which feature is generally accepted as being basic to the automaticity of the sinus node, was inversely related to the volume percentage of myofilaments. This means that a relation exists between the pacemaker action and the cell type. The anatomically less developed cells, i.e. the cells with the lowest density of organelles, which are located in the central portion of the node, are the most specialized pacemakers. No clear relation was found between the myofilament density and the rate of rise of the action potential. In the direction of the crista terminalis we observed an increase in the rate of rise and an increase in conduction velocity concomitant with the increase in myofilament density. Toward the interatrial septum, however, the increase in myofilament density was not accompanied by an increase in rate of rise; in this direction the impulse conduction was blocked. A correlation between cell type and impulse conduction could thus not be established.

Monomeric IgG in intravenous Ig preparations is a functional antagonist of Fc gamma RII and Fc gamma RIIIb

Intravenous Ig preparations (IVIg), originally developed as a substitution therapy for patients with low plasma IgG, are nowadays frequently used in the treatment of various immune diseases. However, the mechanism of action of IVIg in these diseases remains elusive and is often referred to as “immunomodulatory.” We hypothesized that monomeric IgG may act as a low-affinity FcγR antagonist and sought experimental evidence for this hypothesis. Human neutrophils as well FcγRIIa-transfected IIA1.6 cells were used as FcγR-positive cells and aggregated IgG (aIgG) or stable dimeric IgG as FcγR-specific agonists for these cells. We found that monomeric IgG purified from IVIg at concentrations similar to that of IgG in plasma, diminished the binding of stable dimeric IgG to FcγRIIa transfectants, reduced aIgG-induced influx of Ca2+ ions into the cytosol of neutrophils, and attenuated the aIgG-induced release of elastase. Notably, monomeric IgG by itself did not elicit these responses, nor did it affect these processes in response to fMLP. Absorption of IgG from normal plasma revealed that plasma IgG exerted similar effects as monomeric IgG in IVIg. In addition, adding monomeric IgG to blood of healthy volunteers showed a dose-dependent decrease of aIgG-induced elastase release. Finally, we observed decreased aIgG-induced polymorphonuclear neutrophil responses in two hypogammaglobulinemic patients upon treatment with IVIg. We conclude that monomeric IgG at physiological levels acts as a low-affinity FcγR antagonist. Moreover, FcγR antagonism constitutes an immunomodulatory effect of IVIg.

Sinus node and atrium cells from the rabbit heart: A quantitative electron microscopic description after electrophysiological localization

Abstract Electrophysiologically identified cell groups in the sinus node from the rabbit have been compared with atrial fibers with the electron microscope. The point counting method has been used to estimate the volume density of the following structures: nucleus, mitochondria, myofilaments, sarcoplasmic reticulum tubules and subsarcolemmal vesicles. These data were collected in leading pacemaker cells, latent pacemaker cells and atrium cells from the crista terminalis. It has been found that organized structures in leading pacemaker cells occupy about 50% of the cell volume, as compared with over 90% in atrial fibers. Leading pacemaker cells consequently appear extremely “empty”. It has also been found that the group of cells which show the characteristic features of leading pacemaker cells at the ultrastructural level as observed in the correlated experiments is larger than the leading center found in electrophysiology and thus it seems impossible with the actual observation methods to delineate the leading pacemaker center using only cytological criteria.

Rat C-reactive protein activates the autologous complement system

Activation of complement is a biological function of human C‐reactive protein (hCRP), whereas rat CRP (rCRP) has been claimed to be unable to activate complement. As important biological functions of proteins are probably conserved among species, we re‐evaluated, using various ligands, the capability of rCRP to activate complement. The activation of complement by hCRP and rCRP was investigated in solid‐ and fluid‐phase systems. In the solid‐phase system, purified CRP was fixed to enzyme‐linked immunosorbent assay (ELISA) plates and incubated with human or rat recalcified plasma. Dose‐dependent binding of human and rat C3 and C4 was observed to human and rat CRP, respectively. In the fluid‐phase system, recalcified rat plasma, which contains about 500 mg/l of CRP, or human plasma supplemented with hCRP, were incubated with lyso‐phosphatidylcholine. A dose‐dependent activation of complement was observed upon incubation with this ligand, as reflected by the generation of activated C4 as well as of CRP–complement complexes. This activation was, in both cases, inhibited by preincubation of plasma with p‐aminophosphorylcholine, a specific inhibitor of the interaction of CRP with its ligands, or by chelation of calcium ions. We conclude that rat CRP, similarly to human CRP, can activate autologous complement. These results support the notion that opsonization of ligands with complement is an important biological function of CRP.

Asymmetry of the sino-atrial conduction in the rabbit heart

Abstract In isolated right atria of the rabbit heart the impulse conduction from the sinus node to the interatrial septum was studied, using the microelectrode technique. After the electrophysiological experiments several preparations were subjected to a correlative morphological investigation, using light or electron microscopy. By making incisions in the preparation we could demonstrate that the pacemaker region was bordered at the medial (septal) side by a zone in which conduction was blocked completely; the entering wavefronts died out gradually in both antegrade and retrograde directions. The double component action potentials that can be recorded in this region in the spontaneously beating intact preparation, appeared to be an almost passive summation of the wavefronts that entered this zone from the pacemaker side (first component) and from the septal side (second component). The tissue at the septal side was activated by a front that encircled the conduction block. Microscopically the area of the block was not uniform: it showed transition in cell type and tissue architecture, as was observed in a previous study also at the lateral (cristal) border of the node, where the pacemaker impulse is conducted with increasing speed toward the atrium. Neither did we find a difference in spread of electrotonus between the medial and lateral border of the sinus node. The block is therefore thought to be caused by the electrophysiological properties of the cells, most likely their low excitability.

The potentiation of human C1-inhibitor by dextran sulphate is transient in vivo: studies in a rat model.

C1-inhibitor (C1-Inh) is an important regulator of inflammatory reactions because it is a potent inhibitor of the contact and complement system. C1-Inh application in inflammatory disease is, however, restricted because of the high doses required. The glycosaminoglycan-like molecule dextran sulphate (DXS) enhances C1-Inh function in vitro. Hence, we investigated whether co-administration with dextran sulphate reduces the amount of C1-Inh required, through enhancement in vivo. C1-Inh potentiation was measured in a newly developed C1s-inactivation assay that is based on activation of C4 by purified C1s. Activated C4 in rat plasma was quantified with a newly developed ELISA. Human C1-Inh (2.5 microM) inhibited C1s in rat plasma 55-fold faster in the presence of dextran sulphate (15 kDa, 5 microM). To study the stability of the complex in vivo, rats were given a mixture of C1-Inh (10 mg/kg) and dextran sulphate (3 mg/kg). C1-Inh activity during 5 h was analyzed ex vivo with the C1s inactivation assay. The noncovalent C1-Inh-dextran sulphate complex resulted in a transient enhancement of the inhibitory capacity of C1-Inh, lasting for 60-90 min. Dextran sulphate did not affect plasma clearance of C1-Inh. We conclude that the enhanced inhibitory capacity of C1-Inh complexed to dextran sulphate is transient in vivo. Hence, co-administration of these compounds seems a feasible approach to achieve short-term inhibition of complement in vivo.

Intravenous immunoglobulin preparations induce mild activation of neutrophils in vivo via triggering of macrophages studies in a rat model

Despite widespread use in various immune disorders, the in vivo mechanisms of action of intravenous immunoglobulin (IVIG) preparations are not well known. We previously reported that human neutrophils degranulate after incubation with IVIG in vitro as a result of interaction with FcγRII. The purpose of this study was to determine whether IVIG might stimulate neutrophils in vivo. Anaesthetized rats received a bolus intravenous injection of IVIG preparations, containing either high (aged IVIG) or low (fresh IVIG) amounts or IgG dimers at a dose of 250 mg/kg. Administration of aged IVIG induced neutrophil activation in vivo, whereas no effect was observed after infusion of fresh IVIG. Histological examination of lung tissue demonstrated mild influx of neutrophils into the pulmonary tissue after aged IVIG administration, though gross damage did not occur. Macrophage‐depleted rats no longer showed activation of neutrophils after infusion of aged IVIG, suggesting that neutrophils become activated via an indirect macrophage dependent way. We conclude that IVIG induces a mild activation of neutrophils in vivo via triggering of macrophages depending on the amount of IgG dimers. For this reason, IVIG preparations with a high content of dimers may not always be as harmless as generally believed and may be responsible for some of the side‐effects observed during IVIG infusions.

Ultrastructural and Functional Aspects of the Rabbit Sinoatrial Node

The heart beat originates within a certain group of cells of the sinoatrial node (SA node), and from this location, the excitation is propagated through the entire node, the right atrium and the other parts of the heart. Using intracellular recordings of electrical activity, West showed already in 1955 that within small areas of the SA node large differences in the shape of the recorded action potentials could be found. In addition it is known that the leading pacemaker site is small (for review see Brooks and Lu, 1972) and that its location within the node is variable even in animals from the same species (Sano and Yamagishi, 1965; Bleeker et al., 1978). Also morphologically the SA node is not homogeneous while a gradual transition between different territories is observed within the node. So it is difficult to localize precisely the leading pacemaker center. Nevertheless, among the numerous studies dealing with the ultrastructure of the SA node of several mammalian species: mole (Kikuchi, 1976), rat (Bompiani et al., 1959; Viregh, and Porte, 1960; Cheng, 1971), rabbit (Tori, 1962; Trautwein and Uchizono, 1963; Challice, 1966; Tranum-Jensen, 1976), dog (Kawamura, 1961; James et al., 1966; Hayashi, 1971), cow (Rhodin et al., 1961; Hayashi, 1962), monkey (Colborn and Carsey, 1972; Viragh and Porte, 1973) and man (James et al., 1966), only that of Trautwein and Uchizono (1963) has been made after electrophysiological identification, but they selected rather large pieces (1 mm2) for morphological examination.

Temperature dependence of the chronotropic action of calcium: Functional inhomogeneity of the rabbit sinus node

Abstract The chronotropic effect of variation of the external calcium concentration was investigated in the isolated rabbit sinus node at different temperatures. This Ca effect was temperature dependent; at 38°C excess calcium induced an acceleration of the sinus node whereas at 30°C there was on average no significant chronotropic Ca effect. This temperature dependency reconciles the different observations about the chronotropic action of calcium reported by others. With intracellular micro-electrodes the Ca effects were investigated in pacemaking cells of the sinus node. Maps of the activation pattern within the node at different Ca concentrations showed a pacemaker shift from the cranial towards the caudal portion of the node induced by a decrease of the Ca concentration from 2.2 to 1.1 m m . In a concomitant morphological study the intranodal location of the pacemaker centres was confirmed. In the centre leading at 2.2 m m Ca (and higher Ca concentrations) excess calcium increased the rate of diastolic depolarization whereas in the caudal pacemaker centre, leading at 1.1 m m Ca, excess calcium did not change the diastolic depolarization phase. Lowering the temperature induced a pacemaker shift towards the caudal portion of the node also. At 30°C no Ca effect on diastolic depolarization was observed in the leading pacemaker cells. We conclude that the diminished chronotropic action of calcium at low temperature is explained by a temperature dependent pacemaker shift towards cells which are less sensitive to variation of the Ca concentration.