Lipopolysaccharide binding protein derivatives

Details Lipopolysaccharide binding protein derivatives Lipopolysaccharide binding protein derivatives

1999-03-29

2002-04-23

Romeo, David S. (Department: 1646)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S350000, C536S023400, C435S069700, C435S320100, C435S325000, C435S252300

Reexamination Certificate

active

06376462

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates generally to proteins useful for the treatment of gram-negative bacterial infections and specifically to the neutralization of the effects of lipopolysaccharide (LPS) which is also known as endotoxin. LPS is a major component of the outer membrane of gram-negative bacteria and consists of serotype-specific O-side chain polysaccharides linked to a conserved region of core oligosaccharide and lipid A. LPS is an important mediator in the pathogenesis of septic shock and is one of the major causes of death in intensive-care units in the United States. It has been observed that exposure to LPS during sepsis stimulates an immune response in monocytes and macrophages that results in a toxic cascade resulting in the production of tumor necrosis factor (TNF) and other proinflammatory cytokines. Morrison and Ulevitch,
Am. J. Pathol.,
93:527 (1978). Endothelial damage in sepsis probably results from persistent and repetitive inflammatory insults. Bone,
Annals Int. Med.
115:457 (1991).
LPS-binding proteins have been identified in various mammalian tissues. Among the most extensively studied of the LPS-binding proteins is bactericidal/permeability-increasing protein (BPI), a basic protein found in the azurophilic granules of polymorphonuclear leukocytes. Human BPI protein has been isolated from polymorphonuclear neutrophils (PMNs) by acid extraction combined with either ion exchange chromatography or
E. coli
affinity chromatography. Weiss et al.,
J. Biol. Chem.,
253:2664 (1978); Mannion et al.,
J. Immunol.
142:2807 (1989).
The holo-BPI protein isolated from human PMNs has potent bactericidal activity against a broad spectrum of gram-negative bacteria. This antibacterial activity appears to be associated with the amino terminal region (i.e. amino acid residues 1-199) of the isolated human holo-BPI protein. In contrast, the C-terminal region (i.e. amino acid residues 200-456) of the isolated holo-BPI protein displays only slightly detectable anti-bacterial activity. Ooi et al.,
J. Exp. Med.,
174:649 (1991). Human DNA encoding BPI has been cloned and the amino acid sequence of the encoded protein has been elucidated. Gray et al.,
J. Biol. Chem.,
264:9505 (1989). Amino-terminal fragments of BPI include a natural 25 Kd fragment and a recombinant 23 Kd, 199 amino acid residue amino-terminal fragment of the human BPI holoprotein referred to as rBPI
23
. See, Gazzano-Santoro et al.,
Infect. Immun.
60:4754-4761 (1992). In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI
23
) having the 31-residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in SEQ ID NOS: 11 and 12 taken from Gray et al., supra, except that valine at position 151 is specified by GTG rather than GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG). Recombinant holoprotein referred to herein as rBPI has also been produced having the sequence set out in SEQ ID NOS: 11 and 12 taken from Gray et al., supra, with the exceptions noted for rBPI
23
. See also, Elsbach et al., U.S. Pat. No. 5,198,541 the disclosure of which is hereby incorporated by reference. In addition to its bactericidal effects, BPI has been shown to neutralize the toxic and cytokine-inducing effects of LPS to which it binds.
Lipopolysaccharide binding protein (LBP) is a 60 kD glycoprotein synthesized in the liver which shows significant structural homology with BPI. Schumann et al. disclose the amino acid sequences and encoding cDNA of both human and rabbit LBP. Like BPI, LBP has a binding site for lipid A and binds to the LPS from rough (R-) and smooth (S-) form bacteria. Unlike BPI, LBP does not possess significant bactericidal activity, and it enhances (rather than inhibits) LPS-induced TNF production. Schumann et al.,
Science,
249:1429 (1990). Thus, in contrast to BPI, LBP has been recognized as an immunostimulatory molecule. See, e.g., Seilhamer, PCT International Application WO 93/06228 which discloses a variant form of LBP which it terms LBP-&bgr;.
One of the normal host effector mechanisms for clearance of bacteria involves the binding to and subsequent phagocytosis by neutrophils and monocytes. As part of this process, bacteria are exposed to bactericidal and bacteriostatic factors, including oxygen radicals, lysosomal enzymes, lactoferrin and various cationic proteins. LBP opsonizes LPS-bearing particles and intact Gram-negative bacteria, mediating attachment of these LBP-coated particles to macrophages. Wright et al.,
J. Exp. Med.
170:1231 (1989). The attachment appears to be through the CD14 receptor of monocytes which binds complexes of LPS and LBP. Wright et al.,
Science
249:1431 (1990). Anti-CD14 mAbs have been shown to block the synthesis of TNF by whole blood incubated with LPS. Wright et al.
Science
249:1431 (1990). Interaction of CD14, which is present on the surface of polymorphonuclear leukocytes as well as monocytes, with LPS in the presence of LBP has been shown to increase the adhesive activity of neutrophils. Wright et al.,
J. Exp. Med.
173:1281 (1991), Worthen et al.,
J. Clin. Invest.
90:2526 (1992). Thus, while BPI has been shown to be cytotoxic to bacteria and to inhibit proflammatory cytokine production stimulated by bacteria, LBP promotes bacterial binding to and activation of monocytes through a CD14-dependent mechanism.
LPS, either directly or by inducing proinflammatory cytokines such as IL-1 and TNF, induces the expresion of adhesion molecules including CD54 (intercellular adhesion molecule-1, ICAM-1) and E-selectin (endothelial-leukocyte adhesion molecule-1, ELAM-1) on endothelial cells, and thereby increases binding of leukocytes in vitro. Schleimer and Rutledge,
J. Immunol.
136:649 (1986); Pohlman et al.,
J. Immunol.
136:4548 (1986); Bevilacqua et al.,
J. Clin. Invest.
76:2003 (1985); Gamble et al.,
Proc. Natl. Acad. Sci. USA.
82:8667 (1985); Smith et al.,
J. Clin. Invest.
82:1746 (1988); and Bevilacqua et al.,
Proc. Natl. Sci. USA
84:9238 (1987). However, as CD14 has not been detected on the surface of endothelial cells (Beekhuizen et al.,
J. Immunol.
147:3761 (1990)) and no other receptor for LPS on endothelial cells has been identified, a different mechanism may exist whereby LPS can affect the endothelium.
Soluble CD14, found in serum (Bazil et al.,
Eur. J. Immunol.
16:1583 (1986)), has been hypothesized to be responsible for transmitting the LPS signal to endothelial cells. Specifically, soluble CD14 has been shown to mediate a number of LPS-dependent effects on endothelial cells, including E-selectin and VCAM expression, IL-1, IL-6 and IL-8 secretion, and cell death. Frey et al.,
J. Exp. Med.
176:1665 (1992): Pugin et al.,
Proc. Natl. Acad. Sci. USA.
90:2744 (1993).
Recent studies have shown that soluble CD14 is involved in the LPS-mediated adhesion of neutrophils to endothelial cells. Anti-CD14 mAbs were able to completely inhibit the adhesion induced by LPS, indicating that the contribution of other CD14-independent LPS receptors to these effects is minimal. The protein(s) on the endothelial cells that soluble CD14 might associate with to transduce the LPS signal remains to be identified. LBP has been shown to be involved in the signal transduction of LPS through soluble CD14; however, at high concentrations of LPS or soluble CD14, LBP does not further enhance the response of endothelial cells to LPS (Pugin et al.,
Proc. Natl. Acad. Sci. USA.
90:2744 (1993).
Larrick et al.,
Biochem. and Biophysical Res. Commun.,
179:170 (1991) relates to a cationic protein obtained from rabbit granulocytes which is identified as CAP18. CAP18 is identified as bearing no sequence homology with either BPI or LBP. In the course of their disclosure, Larrick et al. characterize other publications which discuss the structure of proteins including LBP and incorrectly attribute to the Wright et al., supra disclosure the speculation that “LBP is believed to be composed of two regions

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