Targeting of Pseudomonas aeruginosa in the Bloodstream with Bispecific Monoclonal Antibodies

Abstract

We examined the ability of a bispecific mAb reagent, consisting of a mAb specific for the primate erythrocyte complement receptor cross-linked with an anti-bacterial mAb, to target bacteria in the bloodstream in an acute infusion model in monkeys. In vitro studies demonstrated a variable level of complement-mediated binding (immune adherence) of Pseudomonas aeruginosa (strain PAO1) to primate E in serum. In vivo experiments in animals depleted of complement revealed that binding of bacteria to E was <1% before administration of the bispecific reagent, but within 5 min of its infusion, >99% of the bacteria bound to E. In complement-replete monkeys, a variable fraction of infused bacteria bound to E. This finding may have significant implications in the interpretation of animal models and in the understanding of bacteremias in humans. Treatment of these complement-replete monkeys with the bispecific reagent led to >99% binding of bacteria to E. Twenty-four-hour survival studies were conducted; several clinical parameters, including the degree of lung damage, cytokine levels, and liver enzymes in the circulation, indicate that the bispecific mAb reagent provides a degree of protection against the bacterial challenge.

Medical biofilms

“…The Taylor group, in series of elegant papers, reports the use of several different bi-specific fusion proteins (BiFPs) that enhanced phagocytosis by macrophage of various pathogens, including: E. coli (Kuhn et al, 1998), P. aeruginosa (Lindorfer et al, 2001), and S. aureus (Gyimesi et al, 2004). In all cases, BiFPs consisted of (1) a molecule that recognizes a surface marker on the pathogen that was chemically coupled with (2) a Mab that is specific to the complement receptor 1 (CR1) present on primate erythrocytes.…”

Analyses of the In Vivo Trafficking of Stoichiometric Doses of an Anti-Complement Receptor 1/2 Monoclonal Antibody Infused Intravenously in Mice

“…Gel filtration analyses (data not shown) revealed that mAbs prepared by this procedure are Ͼ98% monomeric after purification. mAb 7B7 (IgG2a), specific for bacteriophage ⌽X174, has been previously described (36), and was covalently crosslinked to mAb 7G6 using published procedures (37,38). mAbs were labeled with 125 I using Iodogen (39), with Alexa fluorophore (Al) dyes (Al488, Al594, or Al633; Molecular Probes, Eugene, OR) following the manufacturer's directions, or were conjugated with NHS LC biotin (bt; Pierce, Rockford, IL) (40).…”

Staphylococcus aureus bound to complement receptor 1 on human erythrocytes by bispecific monoclonal antibodies is phagocytosed by acceptor macrophages

“…We have found that by constructing bispecific mAb complexes (HP), it is possible to use the anti-CR1 mAb as a surrogate for C3b to facilitate binding of particulate pathogens to E, independent of complement opsonization [7,9,17]. The purpose of the present experiments was to determine whether a clinically relevant strain of S. aureus, bound to human E via HP, could be recognized, removed from E, internalized and killed by acceptor macrophages.…”

“…We prepared HP by coupling mAb 072 with anti-CR1 mAb 9H3 (IgG1 isotype) using previously published methods [9,12]. Biotinylated mAb 012, which is specific for a different site on the T5 isolate, was used for identifying bacteria internalized by macrophages.…”

“…Once bound to E CR1, the HP-pathogen complexes should be recognized by Fc receptors on phagocytic cells in the liver and spleen, leading to removal of the substrates from the circulation followed by their phagocytosis and destruction, without loss of E [7]. Previously, we reported the use of HP to bind two Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to primate E and, in the case of P. aeruginosa, to facilitate safe clearance of this pathogen from the bloodstream of non-human primates [9,10].…”