Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation.

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits. Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity.

Complete mutagenesis of the extracellular domain of interleukin-8 (IL-8) type A receptor identifies charged residues mediating IL-8 binding and signal transduction.

We systematically converted each of the amino acids in the extracellular domain of the interleukin-8 (IL-8) type A receptor to alanine for the purpose of identifying amino acids contributing to IL-8 binding and IL-8-mediated signal transduction. We identified 20 mutations which cause a decrease in receptor affinity from a Kd of 2 nM to a Kd > or = 25 nM. We then analyzed these receptor mutants for their ability to mobilize intracellular calcium upon stimulation with 10 nM IL-8. The majority of the mutants were able to produce calcium fluxes at levels approximating that of wild-type IL-8 receptor A, with the exception of six mutants (R199A, R203A, C30A, C110A, C187A, and C277A) which showed no significant response. In addition, we performed calcium mobilization experiments to further characterize a series of previously constructed mutants which had only been characterized by their binding affinities in our previous report and found that mutant D265A showed no response upon stimulation with 10 nM IL-8. Our study shows that, besides the extracellular domain cysteines which may be critical for the overall folding of the receptor, three residues, Arg-199, Arg-203, and Asp-265, are important for IL-8 binding and IL-8-mediated signal transduction.

Bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. LPS binding properties and effects on LPS-mediated cell activation.

We have previously shown that human bactericidal/permeability-increasing protein (BPI) is able to inhibit serum-dependent lipopolysaccharide (LPS)-mediated activation of human monocytes and neutrophils in vitro, and to counteract the lethal effects of LPS challenge in vivo. Lipopolysaccharide-binding protein (LBP) is a serum protein which participates in LPS-mediated activation of cells (Tobias, P. S., Mathison, J., Mintz, D., Lee, J. D., Kravchenko, V., Kato, K., Pugin, J., and Ulevitch, R. J. (1992) Am. J. Respir. Cell. Mol. Biol. 7, 239-245). We have proposed that BPI functions in a negative feedback loop which opposes this activation (Marra, M. N., Wilde, C. G., Collins, M. S., Snable, J. L., Thornton, M. B., and Scott, R. W. (1992) J. Immunol. 148, 532-537). We have now cloned and expressed recombinant forms of human BPI and LBP. Here we demonstrate that purified recombinant human LBP can replace the serum requirement for both LPS binding to human monocytes and LPS-mediated secretion of tumor necrosis factor alpha from these cells. These activities of LBP are inhibited by a neutralizing anti-CD14 monoclonal antibody. We further demonstrate that purified recombinant human BPI can inhibit LBP-mediated LPS binding to cells and their subsequent activation. Comparison of the LPS binding properties of BPI and LBP in enzyme-linked immunosorbent type assays and in the Limulus amebocyte lysate assay suggest that BPI has a stronger affinity for LPS than does LBP. Direct competition between BPI and LBP for LPS may explain the inhibition by BPI of the proinflammatory effects of LBP in the presence of LPS.

Structure and function of lipopolysaccharide binding protein.

The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

Cloning and expression of the cDNA for bovine granulocyte-macrophage colony-stimulating factor.

A sequence encoding bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified from a concanavalin A-stimulated bovine lymphocyte cDNA library. This sequence was isolated by hybridization with synthetic oligonucleotide probes based upon the human GM-CSF sequence. This bovine cDNA was engineered for expression and secretion of activity into the periplasmic space of E. coli. Periplasmic extracts contain a 14,500-dalton protein and stimulate colony formation of bovine bone marrow progenitor cells. The predicted protein is 70% homologous with human GM-CSF and 55% homologous with murine GM-CSF. Numerous structural features are conserved among these three proteins, such as location of cysteine residues, glycosylation sites, and overall change. The biological activity of bovine GM-CSF is species specific, since recombinant preparations do not cause proliferation of human or murine bone marrow cells. Similarly, murine GM-CSF does not exhibit activity on cells of bovine or human origin. However, human GM-CSF does stimulate colony formation of bovine bone marrow cells, although the specific activity appears reduced when compared to assays on human cells.

Cloning of the cDNA of a human neutrophil bactericidal protein. Structural and functional correlations.

The bactericidal permeability increasing protein (BPI) is a 50-60-kDa membrane-associated protein isolated from granules of polymorphonuclear leukocytes. A full-length cDNA clone encoding human BPI has been isolated and the derived amino acid sequence reveals a structure that is consistent with previously determined biological properties. BPI may be organized into two domains: the amino-terminal half, previously shown to contain all known antimicrobial activity, contains a large fraction of basic and hydrophilic residues. In contrast, the carboxyl-terminal half contains more acidic than basic residues and includes several potential transmembrane regions which may anchor the holoprotein in the granule membrane. The cytotoxic action of BPI is limited to many species of Gram-negative bacteria; this specificity may be explained by a strong affinity of the very basic aminoterminal half for the negatively charged lipopolysaccharides that are unique to the Gram-negative bacterial envelope. The amino-terminal end of BPI exhibits significant similarity with the sequence of a rabbit lipopolysaccharide-binding protein, suggesting that both molecules share a similar structure for binding lipopolysaccharides.