Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-05-19
2001-05-15
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S133100, C424S136100, C424S184100, C435S007100, C435S007900, C435S007920, C435S252300, C435S326000, C435S340000, C436S172000, C436S501000, C436S518000, C436S526000, C436S804000, C530S350000, C530S387100, C530S388100, C530S388230, C530S388250, C530S388400
Reexamination Certificate
active
06232080
ABSTRACT:
TECHNICAL FIELD
The-present invention relates to monoclonal antibodies immunoreactive with lipopolysaccharide binding protein (LBP) and methods for using the antibodies. In addition, the invention relates to methods for detecting the presence of LBP in samples, and to therapeutic methods of using the antibodies for inhibiting LBP-mediated activation of cell of monocyte and macrophage lineage.
BACKGROUND
Sepsis is a morbid condition induced by a toxin, the introduction or accumulation of which is most commonly caused by infection or trauma. The initial symptoms of sepsis typically include chills, profuse sweat, irregularly remittent fever, prostration and the like, followed by persistent fever, hypotension leading to shock, neutropenia, leukopenia, disseminated intravascular coagulation, adult respiratory distress syndrome and multiple organ failure.
Sepsis-inducing toxins have been found associated with pathogenic bacteria, viruses, plants and venoms. Among the well described bacterial toxins are the endotoxins or lipopolysaccharides (LPS) of the gram-negative bacteria. These molecules are glycolipids that are ubiquitous in the outer membrane of all gram-negative bacteria. While chemical structure of most of the LPS molecule is complex and diverse, a common feature is the lipid A region of LPS [Rietschel, E. Th. et al., in
Handbook of Endotoxins,
1:187-214 eds. R. A. Proctor and E. Th. Rietschel, Elsevier, Amsterdam (1984)]; recognition of lipid A in biologic systems initiates many, if not all, of the pathophysiologic changes of sepsis. Because lipid A structure is highly conserved among all types of gram-negative organisms, common pathophysiologic changes characterize gram-negative sepsis.
Current concepts support the contention that the primary response of the host to LPS (including man) involves the recognition of LPS by cells of the monocyte/macrophage lineage, followed by the rapid elaboration of a variety of cell products including the general group known as cytokines. Other cell types believed to participate in sepsis and in particular in the response to LPS are polymorphonuclear leukocytes and endothelial cells; each of these cell types are also capable of responding to LPS with the elaboration of potent inflammatory substances.
LPS is believed to be a primary cause of death in humans during gram-negative sepsis, particularly when the symptoms include adult respiratory distress syndrome (ARDS). van Deventer et al.,
Lancet,
1:605 (1988); Ziegler et al.,
J. Infect. Dis.,
136:19-28 (1987). For instance, one particular cytokine, tumor necrosis factor alpha/cachectin (TNF), has recently been reported to be a primary mediator of septic shock. Beutler et al.,
N. Eng. J. Med.,
316:379 (1987). Intravenous injection of LPS endotoxin from bacteria into experimental animals and man produces a rapid, transient release of TNF. Beutler et. al.,
J. Immunol.,
135:3972(1985). Mathison et al.,
J. Clin. Invest.
81: 1925 (1988). Evidence that TNF is a critical mediator of septic shock comes primarily from experiments in which pretreatment of animals with anti-TNF antibodies reduces lethality. Beutler et al.,
Science,
229:869, (1985). Mathison et al.,
J. Clin. Invest.
81: 1925 (1988). These reports suggest that interruption of the secretion of TNF caused by LPS or other factors would ameliorate the often lethal symptoms of sepsis.
Upon introduction of LPS into the blood, LPS binds to a protein termed lipopolysaccharide binding protein (LBP). LBP is a 60 kD glycoprotein present at concentrations of less than 100 ng/ml in the serum of healthy animals and man. During the acute phase, LBP is synthesized by hepatocytes, and reaches concentrations of 30-50 ug/ml in serum. LBP can be purified from acute phase human and rabbit serum. Tobias, et al.,
J. Exp. Med.,
164:777-793 (1986). LBP recognizes the lipid A region of LPS and forms high affinity, 1:1 stoichiometric complexes with both rough and smooth form LPS. Tobias, et al.,
J. Biol. Chem.,
264:10867-10871 (1989). LBP bears N-terminal sequence homology with the LPS-binding protein known as bactericidal permeability-increasing factor, (BPI). Tobias, et al.,
J. Biol. Chem.,
263:13479-13481, (1988) BPI is stored in the specific granules of PMN [Weiss, et al.,
Blood,
69:652-659, (1987)] and kills gram negative bacteria by binding LPS and disrupting the permeability barrier. Weiss, et al.,
J. Immunol.,
132:3109-3115, (1984). In contrast to BPI, LBP is not directly cytotoxic for gram-negative bacteria [Tobias, et al.,
J. Biol. Chem.,
263:13479-13481, (1988)].
The macrophage/polymorphonuclear leukocyte differentiation antigen, CD14, binds LPS in the presence of LBP when present as LPS-LBP complexes, and this binding event activates cellular responses. Wright et al.,
Science,
249:1431-1433 (1990); Lee et al.,
J. Ex. Med.,
175:1697-1705 (1992). Although it is believed that LPS-LBP binding to CD14 is the important event in LPS-induced activation of myeloid cell lines, it remains unclear what parameters influence LBP-dependent binding of LPS by CD14, and particularly what stoichiometric parameters are involved in the LBP-dependent CD14:LPS interaction.
Monoclonal antibodies immunoreactive with LBP have not been described that interfere with LPS:CD14-mediated cell activation. Therefore, there continues to be a need for reagents that interact with LBP for elucidation and intervention of LBP function.
BRIEF DESCRIPTION OF THE INVENTION
The present invention was born out of the discovery that monoclonal antibodies immunoreactive with LBP can interfere with LPS:CD14 function, and particularly that inhibit LPS binding to CD14. Surprisingly, anti-LBP antibodies are described that do not inhibit LPS binding to LBP, and yet interfere with LPS:CD14 binding.
Thus, in one embodiment the invention contemplates an monoclonal antibody that immunoreacts with lipopolysaccharide (LPS) binding protein (LBP) but does not substantially inhibit LBP binding to LPS. Preferably, the LBP is human LBP.
Preferably, the monoclonal antibody also inhibits LBP-mediated binding of LPS to CD14. More preferably, the monoclonal antibody inhibits LBP-mediated LPS-dependent activation of myeloid cells, and still more preferably the monoclonal antibody inhibits LBP-mediated LPS-dependent secretion of tumor necrosis factor from myeloid cells.
Also contemplated are hybridoma cell lines that produce a monoclonal antibody of this invention.
In a further embodiment, the invention contemplates therapeutic compositions, typically in unit dose form, useful for inhibiting LPS binding to CD14, and therefore can be used for preventing or ameliorating the symptoms of LPS mediated cell activation, including cytokine production and sepsis. The compositions comprise a pharmaceutically acceptable carrier containing one or more of an anti-LBP antibody that acts as an LBP antagonist, as an active ingredient.
In preferred embodiments, a therapeutic composition of this invention further contains, as active ingredients an agent known to prevent or ameliorate the symptoms of sepsis, such as an antibiotic, steroid, anti-TNF antibody, a TNF antagonist, soluble CD14 and the like, either alone, in sub-combination or combination.
The present invention also contemplates administering, preferably intravenously, to a patient at risk for or suffering the symptoms of LBP-mediated LPS-dependent cell activation a therapeutically effective amount of an anti-LBP antibody. The method can be practiced alone or in combination with the substantially simultaneous administration of other therapeutic modalities known to prevent or ameliorate the symptoms of LBP-mediated LPS-dependent cell activation, including treatment with one or more of an antibiotic, steroids, anti-TNF antibody, TNF antagonist and the like.
Diagnostic methods for detecting LBP in a sample using one or more anti-LBP-antibodies are also contemplated as are kits useful for the detection of LBP containing anti-LBP antibodies.
A major advantage of the present invention lies in the discovery that the monoclona
Kirkland Theo
Leturcq Didier
Moriarty Ann
Tobias Peter
Ulevitch Richard
Fitting Thomas
Hines Ja-Na A.
Northrup Thomas E.
Smith Lynette R. F.
The Scripps Research Institute
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