Recombinant endotoxin-neutralizing proteins

Details Recombinant endotoxin-neutralizing proteins Recombinant endotoxin-neutralizing proteins

1995-05-01

2001-07-24

Housel, James (Department: 1645)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S320100, C424S001110, C424S001570, C536S023100

Reexamination Certificate

active

06265187

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to the field of recombinant, endotoxin-neutralizing proteins, particularly to recombinant proteins which bind endotoxin and block endotoxin-mediated activation of biological systems.
BACKGROUND OF THE INVENTION
Gram-negative infections are a major cause of morbidity and mortality, especially in hospitalized and immunocompromised patients. [Duma,
Am. J. of Med.,
78 (Suppl. 6A):154-164 (1985); and Kreger et al.,
Am. J. Med.,
68:344-355 (1980)]. Although available generally effective in inhibiting growth of Gram-negative bacteria, they do not neutralize the pathophysiological effects associated with endotoxins. Endotoxin is a heat stable bacterial toxin composed of lipopolysaccharides (LPS) released from the outer membrane of Gram-negative bacteria upon lysis [Shenep et al.,
J. Infect. Dis.,
150(3):380-388 (1984)], and is a potent stimulator of the inflammatory response. Endotoxemia occurs when endotoxin enters the bloodstream resulting in a dramatic systemic inflammatory response.
Many detrimental in vivo effects of LPS result from soluble mediators released by inflammatory cells. [Morrison et al.,
Am. J. Pathol.,
93(2):527-617 (1978)]. Monocytes and neutrophils, which ingest and kill microorganisms, play a key role in this process. Monocytes and neutrophils respond to endotoxin in vivo by releasing soluble proteins with microbicidal, proteolytic, opsonic, pyrogenic, complement-activating and tissue-damaging effects. These factors mediate many of the pathophysiological effects of endotoxin. For example, tumor necrosis factor (TNF), a cytokine released by endotoxin-stimulated monocytes, causes fever, shock, and alterations in glucose metabolism and is a potent stimulator of neutrophils. Other cytokines such as IL-1, IL-6, and IL-8 also mediate many of the pathophysiologic effects of LPS, as well as other pathways involving endothelial cell activation by tissue factor, kininogen, nitric oxide and complement.
Endotoxin-associated disorders result from extra-gastrointestinal exposure to LPS, e.g. administration of LPS-contaminated fluids, or Gram-negative infections. Endotoxin-associated disorders can also result when the natural epithelial barrier is injured and the normal Gram-negative flora breach this barrier. For example, endotoxin-associated disorders can occur (a) when there is ischemia of the gastrointestinal tract (e.g., following hemorrhagic shock or during certain surgical procedures), or (b) when systemic or local inflammation causes increased permeability of the gut to endotoxin or Gram-negative organisms. The presence of endotoxin and the resulting inflammatory response may result, for example, in endotoxemia, systemic inflammatory response syndrome (SIRS), sepsis syndrome, septic shock, disseminated intravascular coagulation (DIC), adult respiratory distress syndrome (ARDS), cardiac dysfunction, organ failure, liver failure (hepatobiliary dysfunction), brain failure (CNS dysfunction), renal failure, multi-organ failure and shock.
Examples of diseases which can be associated with Gram-negative bacterial infections or endotoxemia include bacterial meningitis, neonatal sepsis, cystic fibrosis, inflammatory bowel disease and liver cirrhosis, Gram-negative pneumonia, Gram-negative abdominal abscess, hemorrhagic shock and disseminated intravascular coagulation. Subjects who are leukopenic or neutropenic, including subjects treated with chemotherapy or immunocompromised subjects (for example with AIDS), are particularly susceptible to bacterial infection and the subsequent effects of endotoxin.
Several therapeutic compounds have been developed to inhibit the toxic effects of endotoxin, including antibacterial LPS-binding agents and anti-LPS antibodies, although each has met with limitations. For example, Polymyxin B (PMB) is a basic polypeptide antibiotic which binds to Lipid A, the most toxic and biologically active component of endotoxin. PMB inhibits endotoxin-mediated activation of neutrophil granule release in vitro and is a potential therapeutic agent for Gram-negative infections. However, because of its systemic toxicity, this antibiotic has limited therapeutic use, and is generally used topically. Combination therapy using antibiotics and high doses of methylprednisolone sodium succinate (MPSS) showed more promise as this regimen prevented death in an experimental animal model of Gram-negative sepsis. However, a clinical study using MPSS with antibiotics in treatment of patients having clinical signs of systemic sepsis showed that mortality rates were not significantly different between the treatment and placebo groups [Bone et al.,
N. Engl. J. Med.
317:653 (1987)].
Antibodies that bind endotoxin have been used in the treatment of endotoxemia. For example, hyperimmune human antisera against
E. coli
J5 reduced mortality by 50% in patients with Gram-negative bacteremia and shock [Ziegler et al.,
N. Engl. J. Med.
307:1225 (1982)]. However, attempts to treat Gram-negative sepsis by administration of anti-LPS monoclonal antibodies met with little or no success [Ziegler et al.,
N. Engl. J. Med.
324:429 (1991); Greenman et al.,
JAMA
266:1097 (1991); Baumgartner et al.,
N. Engl. J. Med.
325:279 (1991)].
Another approach to treating endotoxemia involves the use of cytokine blockers, such as IL-1 receptor antagonists and anti-TNF antibodies, as well as the soluble forms of the IL-1 and TNF receptors. However, any given cytokine blocker blocks only the cytokine for which it is specific, and fails to prevent the action of other cytokines. Furthermore, blocking cytokines may have other deleterious effects.
Two soluble endotoxin-binding proteins, lipopolysaccharide binding protein (LBP) and bactericidal/permeability-increasing (BPI), play opposing roles in vivo in the physiological response to endotoxin. LBP is a soluble LPS receptor found in serum which binds LPS with high affinity via interaction with the Lipid A moiety [Tobias et al. (1986) J. Exp. Med. 164:777-793; Tobias et al. (1989) J. Biol. Chem. 264:10867-10871]. LBP-LPS complexes stimulate monocyte activation through interaction with the CD14 receptor on the surface of monocytes, resulting in production of cytokines such as TNF and IL-1 [Wright et al. (1989) J. Exp. Med. 170:1231-1241; Wright et al. (1990) Science 249:1431]. Thus, LBP acts as a transfer protein in LPS-mediated stimulation of cytokine release. Moreover, LBP increases LPS activity in that a lower concentration of LPS is required to stimulate monocytes in the presence of LBP than in its absence.
In direct contrast to LBP, BPI binds and neutralizes endotoxin, preventing inflammatory cell activation. BPI, also known as CAP57 and BP [Shafer et al.,
Infect. Immun.
45:29 (1984); Hovde et al.,
Infect. Immun.
54:142 (1986)] is also bactericidal by virtue of its interaction with the Lipid A moiety of LPS in the bacterial cell wall. BPI binds LPS, disrupts LPS structure and the cell wall, and increases bacterial membrane permeability, resulting in cell death [Weiss et al., J. Biol. Chem, 253:2664-2672 (1978); Weiss et al., Infection and Immunity 38:1149-1153 (1982)]. BPI retains its in vitro bactericidal activity after protease cleavage, suggesting that BPI fragments retain activity [Ooi et al., Clinical Research 33(2):567A (1985)]. This observation was confirmed by Ooi et al., who showed that an N-terminal 25 kD fragment of BPI exhibited both the in vitro bactericidal and permeability increasing activities [Ooi et al., J. Biol. Chem. 262:14891 (1987)].
Molecular Structures of BPI and LBP
The genes encoding BPI and LBP have been cloned [Gray et al. (1989) J. Biol. Chem. 264:9505-9509; Schumann et al.,
Science
249:1429-1431 (1990)]. BPI and LBP are immunologically cross-reactive, contain a hydrophobic leader sequence, and share significant amino acid sequence homology over the entire length of the molecules, with an overall amino acid sequence identity of 44

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