Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-30
2003-10-07
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S004000, C436S501000, C436S503000, C536S001110, C536S123100, C536S123120, C536S055100
Reexamination Certificate
active
06630310
ABSTRACT:
BACKGROUND OF THE INVENTION
Underivatized, aqueous soluble &bgr;(1-3)-glucan (also known as PGG-Glucan or Betafectin®) is a novel and unique soluble &bgr;-glucan manufactured through a proprietary process. The biological activity of this molecule is clearly distinguishable from particulate or other soluble &bgr;-glucans. Numerous laboratories have reported direct induction of arachidonic acid metabolites (Czop et al.,
J. Immunol
. 141(9):3170-3176 (1988)), cytokines (Abel and Czop,
Intl. J. Immunopharmacol
. 14(8):1363-1373 (1992); Doita et al.,
J. Leuk. Biol
. 14(2):173-183 (1991)) and oxidative burst (Cain et al.,
Complement
, 4:75-86 (1987); Gallin et al.,
Int. J. Immunopharmacol
. 14(2):173-183 (1992)) by both particulate and soluble forms of &bgr;-glucans. In contrast, underivatized, aqueous soluble &bgr;(1-3)-glucan does not directly activate leukocyte functions such as oxidative burst activity (Mackin et al.,
FASEB J
. 8:A216 tokine secretion (Putsiaka et al., Blood 82:3695-3700 (1993)) or proliferation (Wakshull et al.,
J. Cell. Biochem. suppl
. 18A:22 (1994)). Instead, underivatized, aqueous soluble &bgr;(1-3)-glucan primes cells for activation by secondary stimuli (Mackin et al. (1994); Brunke-Reese and Mackin,
FASEB J
. 8:A488 (1994); and Wakshull et al. (1994)).
The biological activity of &bgr;-glucans is mediated through specific receptors located on target cells. Several groups of investigators have described receptors which bind particulate &bgr;-glucan preparations. For example, receptors for particulate &bgr;-glucans (e.g., zymosan-like particles) have been described by Czop and colleagues (Czop and Kay,
J. Exp. Med
. 173:1511-1520 (1991); Szabo et al.,
J. Biol. Chem
. 270:2145-2151 (1995)) and Goldman (
Immunology
63(2):319-324 (1988);
Exp. Cell. Res
. 174(2):481-490 (1988)). The leukocyte complement receptor 3 (CR3, also known as MAC 1 or CD11b/CD18) has been shown to have the capacity to bind both particulate and some soluble &bgr;-glucans, as well as other polysaccharides (Thornton et al.,
J. Immunol
. 156:1235-1246 (1996)). A soluble aminated &bgr;-glucan preparation has been shown to bind to murine peritoneal macrophages (Konopski et al.,
Biochim. Biophys. Acta
1221:61-65 (1994)), and a phosphorylated &bgr;-glucan derivative has been reported to bind to monocyte cell lines (Muller et al.,
J. Immunol
. 156:3418-3425 (1996)). The ability of salmon macrophages (Engstad and Robertsen,
Dev. Comp. Immunol
. 18(5):397-408 (1994)) and brain microglial cells (Muller et al.,
Res. Immunol
. 145:267-275 (1994)) to phagocytose &bgr;-glucan particles, presumably through a receptor-mediated process, has also been described.
Unfortunately, each group has utilized &bgr;-glucan preparations varying widely in their source, method of preparation, purity and characterization. In addition, different cell types and species, both primary and established cell lines, and different functional read-outs have been used. The relationship between the various receptors described by these investigators has, therefore, not been defined, although it is clear that the receptor described by Czop is not CR3 (Szabo et al. (1995)).
SUMMARY OF THE INVENTION
This invention pertains to the discovery that underivatized, aqueous soluble &bgr;(1-3)-glucan specifically binds to a novel receptor located on human leukocyte membranes (HLM). As described herein, a radiolabeled underivatized, aqueous soluble &bgr;(1-3)-glucan was used to measure the binding of this &bgr;-glucan to membrane receptors derived from human leukocytes as well as various murine and human cell lines. The receptor for underivatized, aqueous soluble &bgr;(1-3)-glucan shows specific and saturable binding to membranes and is highly selective for a subclass of soluble &bgr;-glucans. Results of work described herein characterize this receptor for underivatized, aqueous soluble &bgr;(1-3)-glucan and clearly differentiate it from previously described &bgr;-glucan receptors for either particulate or soluble &bgr;-glucans, while revealing important information about the mechanism of underivatized, aqueous soluble &bgr;(1-3)-glucan biological activity.
This invention also pertains to a method of altering (e.g., activating or deactivating) signal transduction pathways, for example through modulation of one or more transcriptional regulatory factors in receptor-positive cells, i.e., cells which contain the receptor for underivatized, aqueous soluble &bgr;(1-3)-glucan. In one embodiment of the invention, the signal transduction pathway is modulated or regulated by one or more transcriptional regulatory factors from the NF-&kgr;B and/or NF-IL6 and/or jun/fos families of transcriptional regulatory factors. For example, the transcriptional regulatory factor can be NF-&kgr;B, NF-IL6 or AP-1.
Other signal transduction pathways which can be altered by the methods of the present invention include the ras/raf-1/MAP kinase pathway, the G-protein/phospholipase C/protein kinase C pathway, the JAK/STAT pathway, the phospholipase A pathway, G-protein/phospholipase D/phosphatidic acid pathway and the c-AMP-dependent pathway. In each pathway, an appropriate activator or indicator of the signal pathway is activated by binding of underivatized, aqueous soluble &bgr;(1-3)-glucan to its receptor, and modulation of this binding can alter the corresponding signal transduction.
According to the method of the present invention, the activity of the receptor for underivatized, aqueous soluble &bgr;(1-3)-glucan is activated through binding of an underivatized, aqueous soluble &bgr;(1-3)-glucan, whereby a signal transduction process is activated such that one or more transcriptional regulatory factors (e.g., from the NF-&kgr;B, NF-IL6 or jun/fos families) are activated. Activation of these transcriptional regulatory factors can be used to measure the activation of the associated signal transduction pathway. Activation of the receptor can comprise, among others, an alteration in the receptor conformation, formation of a ligand-receptor complex, or alteration of the ligand-receptor complex. Alternatively, the activity of the receptor can be initiated by an agent which mimics the binding and activation ability of an underivatized, aqueous soluble &bgr;(1-3)-glucan. In a particular embodiment, the transcriptional regulatory factor is activated as a result of ligand binding. In another embodiment, the activity of the transcriptional regulatory factor is decreased, either partially or totally, by the binding of an agent to the receptor (and thus excludes the underivatized, aqueous soluble &bgr;(1-3)-glucan), but lacks the ability to activate the receptor.
The invention also pertains to an assay for identifying agents which alter (e.g., increase or decrease) the binding of underivatized, aqueous soluble &bgr;(1-3)-glucan to its receptor. The assay comprises combining radiolabeled underivatized, aqueous soluble &bgr;(1-3)-glucan, the receptor for underivatized, aqueous soluble &bgr;(1-3)-glucan, and an agent to be tested, under conditions suitable for binding of underivatized, aqueous soluble &bgr;(1-3)-glucan to its receptor. The extent of binding of underivatized, aqueous soluble &bgr;(1-3)-glucan to its receptor in the presence of the agent to be tested is determined and compared with the extent of binding in the absence of the agent to be tested; a difference in the extent of binding indicates that the agent alters the binding of underivatized, aqueous soluble &bgr;(1-3)-glucan to its receptor. An increase in the extent of binding in the presence of the agent indicates that the agent enhances, i.e., prolongs or increases, binding or is an agonist of the receptor for underivatized, aqueous soluble &bgr;(1-3)-glucan. A decrease in the extent of binding in the presence of the agent indicates that the agent diminishes, i.e., shortens or decreases, binding or is an antagonist of the receptor for underivatized, aqueous soluble &bgr;(1-3)-glucan. The invention also relates to agents identified by assays described herein, and accordingly, relates to agonists and
Mackin William M.
Wakshull Eric
Zimmerman Janet
Biopolymer Engineering Pharmaceutical, Inc.
Darby & Darby
Kaufman Claire M.
Spector Lorraine
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