Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds antigen or epitope whose amino acid sequence is...
Reexamination Certificate
2001-07-12
2004-10-19
Chan, Christina (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds antigen or epitope whose amino acid sequence is...
C424S152100
Reexamination Certificate
active
06805863
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for modulating immune coagulation using novel antibodies and compounds that modulate immune coagulation.
BACKGROUND OF THE INVENTION
Activation of the coagulation pathways is an important part of immune and inflammatory reactions and is associated with bacterial and viral infections (e.g. endotoxin shock, viral hepatitis), glomerulonephritis (GN), cancer, a number of gastrointestinal diseases, allograft and xeno graft rejection and spontaneous or stress-triggered fetal loss. Immune coagulation is mediated by a number of coagulants that, when triggered, activate specific ligands resulting in cleavage and activation of coagulation pathways that lead to fibrin deposition. The molecular events leading to expression of immune coagulants involve natural antibodies binding both to antigens on endothelial cells and Fc receptors on macrophages and endothelial cells. An additional mechanism is immune complex-mediated induction of macrophage procoagulants. These events lead to thrombin production which initiates platelet activation and ultimately fibrin deposition.
In 50% of hepatitis patients moderate to severe consumptive coagulopathy or disseminated intravascular coagulopathy is found associated with fulminant hepatitis. Thrombi formation is observed around necrotic areas (Sinclair et al., 1990 and Lee, W. M., 1993). As a consequence of hepatitis, levels of factors II, V, VII, and X are decreased in the liver, reflecting both consumptive coagulopathy and a decrease in hepatic synthetic function. Also, the levels of thrombin-antithrombin complexes are high and platelet counts are low (Lee, W. M., 1993). These results indicate that the host immune system, including the coagulation pathway, is disrupted as a result of HBV infection. The limited host range of HBV and the difficulty to propagate the virus in tissue culture have hampered the understanding of HBV and hepatitis B.
Mononuclear phagocytes and macrophages are implicated in the pathogenesis of hepatitis specific induction of procoagulant activity because of their role in coagulation; they synthesize some of the essential coagulation factors such as tissue factor and their surfaces serve as sites of fibrin deposition. Factors participating in the coagulation cascade are released as inactive zymogens and upon activation, by preceding activated factors, they are converted to their active form. The factors are predominantly serine proteases (Davie et al., 1991). Factors VIIa, XIIa, XIa, Xa, IXa, thrombin, kallikrein, and plasminogen are categorized under family 1 serine proteases (Davie et al., 1991; Barrett and Rawlings, 1995; Rawlings and Barrett, 1994; Nduwimana et al., 1995). In order to initiate the coagulation cascade the procoagulants need to be expressed. Ruegg and Pytela, 1995 isolated a cDNA encoding a protein that is homologous to a murine fibrinogen-like protein (Koyama et al. 1987). However, they did not determine the function of the protein or realize its use in modulating immune coagulation.
In view of the many diseases associated with the activation of the coagulation pathways, there is a need to identify and characterize procoagulants and to develop methods for modulating immune coagulation that are useful in the prevention, treatment and diagnosis of diseases associated therewith including bacterial and viral infections, glomerulonephritis (GN), cancer, a number of gastrointestinal diseases, allograft and xenograft rejection and spontaneous or stress-triggered fetal loss.
SUMMARY OF THE INVENTION
The present inventor has identified and characterised an immune procoagulant, and the molecular and cellular events leading to its production. Specifically, the mouse and human direct prothrombinase genes (referred to herein as “mFgl2” and “hFgl2” respectively) have been cloned and sequenced. The nucleic acid sequence of the human and mouse Fgl2 is shown in SEQ.ID.NOS.:1 and 3, respectively. The genes encode a transmembrane serine protease which has functional prothrombinase activity. The proteins encoded by the genes have been sequenced in both humans and mice. The protein has a molecular weight of approximately 70 kd. The hfg12 gene has been mapped to chromosome 7 and the mFgl2 gene to chromosome 5. The inventor has cloned and sequenced the genomic DNA encoding the human prothrombinase. The organization of the genomic DNA encoding hFgl2 is shown schematically in FIG.
1
. The nucleic acid sequence of the promoter region, exon 1, exon 2 and the 3′ UTR are shown in
FIGS. 8
,
2
,
3
and
4
, respectively. The amino acid sequence of the human and mouse Fgl2 protein is shown in FIG.
5
and in SEQ.ID.NOS.:2 and 4, respectively.
The determination by the inventor that Fgl2 is a direct prothrombinase allows the development of diagnostic methods and therapies for conditions involving immune coagulation.
Accordingly, the present invention provides a method of inhibiting immune coagulation comprising inhibiting the activity or expression of Fgl2. The method can be used in viva to treat a condition which requires a reduction in immune coagulation such as bacterial and viral infections, glomerulonephritis (GN), cancer, a number of gastrointestinal diseases, allograft and xenograft rejection and fetal loss.
In one aspect, the activity of Fgl2 may be inhibited using an antibody that binds to Fgl2. The present inventor has developed a pariel of monoclonal and polyclonal antibodies which neutralize Fgl2 and prevent the fibrin deposition associated with endotoxin shock, viral hepatitis, allograft and xenograft rejection. The antibodies were shown to prevent cellular infiltration, fibrin deposition and tissue damage, and lead to enhanced survival. In particular, antibodies against the direct prothrombinase (Fgl2) were found to be extremely useful in preventing diseases known to have associated massive fibrin deposition and coagulative necrosis, including allograft and xenograft rejection as well as fetal loss induced by stress or cytokines.
In one embodiment, the present invention provides a method of preventing or reducing graft rejection comprising administering an effective amount of an antibody to Fgl2 to an animal in need thereof.
In another embodiment, the present invention provides a method of preventing or reducing fetal loss comprising administering an effective amount of antibody to Fgl2 to an animal in need thereof.
Antibodies can be prepared using entire Fgl2 proteins or immunogenic portions thereof. Preferably, such portions bind with an affinity of at least about 10
6
L/mole to an antibody raised against Fgl2. In particular, the present inventor has shown that a peptide comprising amino acid residues 300 to 400 is useful in raising antibodies. Accordingly, the present invention contemplates antibodies which (a) immunoreact with peptides comprising the amino acids at approximately positions 300 to 400 in
FIG. 5
; and (b) neutralize the prothrombinase activity of hFgl2. The invention also relates to hybridoma cell lines that produce the monoclonal antibodies, and inhibitors and activators thereof.
In another aspect, the expression of Fgl2 may be inhibited using antisense molecules that are complimentary to a nucleic acid sequence from the Fgl2 gene. In particular, the nucleic acid sequences for Fgl2 as shown in
FIGS. 2
or
3
may be inverted relative to their normal presentation for transcription to produce antisense nucleic acid molecules.
Additional inhibitors of Fgl2 may be identified by testing substances that inhibit the prothrombinase activity of Fgl2. In particular, the invention contemplates a method for assaying for a substance that affects the prothrombinase activity of Fgl2 comprising (a) reacting Fgl2, a substrate which is capable of being cleaved by Fgl2 to produce a product, and a test substance, under conditions which permit cleavage of the substrate to produce the product; (b) assaying for product; and (c) comparing to the product obtained in the absence of the substance to determine the affect of the substance on the prothrombinase act
Bereskin & Parr
Chan Christina
Gravelle Micheline
Haddad Maher
Trillium Therapeutics Inc.
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