AU749716B2

AU749716B2 - Receptor for underivatized, aqueous soluble beta-(1,3)-glucan

Info

Publication number: AU749716B2
Application number: AU56819/99A
Authority: AU
Inventors: David S. Adams; Eric M. Wakshull
Original assignee: Collaborative Group Ltd
Current assignee: Collaborative Group Ltd
Priority date: 1998-08-26
Filing date: 1999-08-19
Publication date: 2002-07-04
Anticipated expiration: 2019-08-19
Also published as: EP1105733A1; MXPA01002022A; CA2341668A1; JP2002523522A; WO2000013019A1; JP2011102323A; AU5681999A; WO2000013019A9

Description

WO 00/13019 PCT/US99/18995 -1- RECEPTOR FOR UNDERIVATIZED, AQUEOUS SOLUBLE BETA-(1.3)-GLUCAN BACKGROUND OF THE INVENTION Underivatized, aqueous soluble P(1,3)-glucan (also known as PGG glucan, triple helix glucan (TH-glucan) or Betafectin®) is a novel and unique soluble Pglucan manufactured through a proprietary process. The biological activity of this molecule is clearly distinguishable from particulate or other soluble P-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 P-glucans. In contrast, underivatized, aqueous soluble P(1,3)-glucan does not directly activate leukocyte functions such as oxidative burst activity (Mackin et al., FASEB J. 8:A216 (1994)), cytokine secretion (Putsiaka et al.,Blood 82:3695-3700 (1993)) or proliferation (Wakshull et al., J 15 Cell. Biochem. suppl. 18A:22 (1994)). Instead, underivatized, aqueous soluble P(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)).

*e The biological activity of P-glucans is mediated through specific receptors located on target cells. Several groups of investigators have described receptors which bind to and mediate phagocytosis of particulate P-glucan preparations zymosan-like particles; Goldman (Immunology 63(2):319-324 (1988); Exp. Cell.

Res. 174(2):481-490 (1988); Engstad and Robertsen, Dev. Comp. Immunol.

WO 00/13019 PCT[US99/18995 -2- 18(5):397-408 (1994); Muller et al., Res. Immunol. 145:267-275 (1994)); Czop, Advances in Immunol. 38:361,398 (1986)); and have partially characterized these receptors (Czop and Kay, J. Exp. Med. 173:1511-1520 (1991); Szabo et al., J. Biol.

Chem. 270:2145-2151 (1995)). The leukocyte complement receptor 3 (CR3, also known as MAC 1 or CD1 Ib/CD18) has been reported to bind both particulate and some soluble P-glucans, as well as other polysaccharides (Thornton et al., J.

Immunol. 156:1235-1246 (1996)). A soluble aminated P-glucan preparation has been shown to bind to murine peritoneal macrophages (Konopski et al., Biochim.

Biophys. Acta 1221:61-65 (1994)), and a phosphorylated P-glucan derivative has been reported to bind to monocyte cell lines (Muller et al., J. Immunol. 156:3418- 3425 (1996)).

Unfortunately, each group has utilized P-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 to characterize the biology and biochemistry of the interations between these P-glucans and the target cells. 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 P(1,3)-glucan specifically activates a novel receptor located on rat NR8383 macrophages. As described herein, a radiolabeled underivatized, aqueous soluble P(1,3)-glucan was used to measure the binding of this P-glucan to membrane receptors derived from rat NR8383 cells. The receptor for underivatized, aqueous soluble P(1,3)-glucan is a protein, and shows specific and saturable binding to membranes and is highly selective for a subclass of soluble P-glucans. Results of work described herein characterize this receptor for underivatized, aqueous soluble P(1,3)-glucan and clearly differentiate it from previously described P-glucan receptors for either particulate or soluble P-glucans, while revealing important WO 00/13019 PCTIUS99/1 8995 -3information about the mechanism of underivatized, aqueous soluble P(1,3)-glucan biological activity.

The present invention relates to isolated preparations containing the receptor described herein. In addition, the present invention pertains to methods of isolating, concentrating or purifying p(1,3)-glucans, as well as p(1,3)-glucan-containing organisms, and also to assays to quantify the amount of 1(1,3)-glucans, or P(1,3)glucan-containing organisms in a test sample. The test sample can be a liquid or a solid, and can originate from various sources, including complex animal sources a biological fluid, such as blood, serum, plasma, urine, feces, mucus, sputum, bile, ascites fluid, wound secretions, vaginal excretions, synovial fluid, cerebrospinal fluid, peritoneal lavage fluid, lung lavage fluid, ocular fluid, saliva or whole tissue extract; alternatively, the test sample can be solid specimen, such as skin or other tissue); plant sources (plant tissue, plant tissue extract, fruit or fruit extracts, seeds or seed extracts, sap, or homogenates); bacterial cells; viral cells; fungal cells; tissue cultures; environmental sources; food sources; or fermentation process sources.

In the methods of the invention, the receptor described herein is used to capture or purify P(1,3)-glucans and/or p(1,3)-glucan-containing organisms from a test sample. The P(1,3)-glucan receptor, or the test sample, can be coupled or attached to a solid phase or a fluid phase. The test sample is contacted with the receptor under conditions that are suitable for binding of any P(1,3)-glucan that may be present in the test sample to the receptor; resultant complexes of P(1,3)-glucan and receptor ("primary complexes") can then be isolated, in order to isolate P(1,3)glucan. In another embodiment, the primary complexes can be detected, in order to detect P(1,3)-glucan or P(1,3)-glucan-containing organisms in the test sample: the presence of primary complexes is indicative of the presence of P(1,3)-glucan or P(1,3)-glucan-containing organisms. In another embodiment, the presence of P(1,3)-glucan can be detected through a competition assay, in which labeled underivatized, aqueous soluble P(1,3)-glucan and receptors are incubated with the test sample; the amount of 1(1,3)-glucan in the test sample is inversely proportional to the amount of complexes of labeled underivatized, aqueous soluble P(1,3)-glucan and receptors. The presence of P(1,3)-glucan (or of p(1,3)-glucan-containing organisms) in the test sample is indicative of a fungal infection or fungal WO 00/13019 PCT/US99/18995 -4contamination. Antibodies that can be used in these methods and assays, as well as kits that can be used in these methods and assays, are also within the scope of the invention.

This invention also pertains to a method of altering activating or deactivating) signal transduction pathways, for example through modulation of one or more transcriptional regulatory factors in receptor-positive cells, cells which contain the receptor for underivatized, aqueous soluble p(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-KB and/or NF-IL6 and/or AP-1 families of transcriptional regulatory factors.

Other signal transduction pathways which can be altered by the methods of the present invention include the ras/raf-1/MAP kinase (ERK) pathway, the Gprotein/phospholipase C/protein kinase C pathway, the non-G-protein/phospholipase C/protein kinase C pathway, the JAK/STAT pathway, the phospholipase A pathway, G-protein/phospholipase D/phosphatidic acid pathway, the c-AMP-dependent pathway, the c-Jun N-terminal kinase (JNK, also known as stress-activated protein kinase (SAPK)) pathway, the IKB kinase a (IKKa) pathway, the protein tyrosine kinase pathway, and the oxygen radical pathway. In each pathway, an appropriate activator or indicator of the signal pathway is activated by binding of underivatized, aqueous soluble P(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 P(1,3)-glucan is activated through binding of an underivatized, aqueous soluble P(1,3)-glucan, or an agent that mimics the activity of underivatized, aqueous soluble P(1,3)-glucan, whereby a signal transduction process is activated such that one or more transcriptional regulatory factors from the NF-KB, NF-IL6 and/or AP-1 families) are activated. Activation of these transcriptional regulatory factors or of the enzymes in the pathway of transcription factor activation (referred to herein as "pathway enzymes") 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.

WO 00/13019 PCT/US99/1 8995 The activity of the receptor can be initiated by an agent which mimics the binding and activation ability of an underivatized, aqueous soluble P(1,3)-glucan, such as another conformer of the glucan. In a particular embodiment, the transcriptional regulatory factor is activated as a result ofligand 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 P(1,3)-glucan), but lacks the ability to activate the receptor.

The invention also pertains to an assay for identifying agents which alter increase or decrease) the binding of underivatized, aqueous soluble P(1,3)glucan to its receptor. The assay comprises combining radiolabeled underivatized, aqueous soluble P(1,3)-glucan, the receptor for underivatized, aqueous soluble P(1,3)-glucan, and an agent to be tested, under conditions suitable for binding of underivatized, aqueous soluble P(1,3)-glucan to its receptor. The extent of binding of underivatized, aqueous soluble p(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 P(1,3)-glucan to its receptor. An increase in the extent of binding in the presence of the agent indicates that the agent enhances, prolongs or increases, binding or is an agonist of the receptor for underivatized, aqueous soluble P(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 p(1,3)-glucan. The invention also relates to agents identified by assays described herein, and accordingly, relates to agonists and antagonists of underivatized, aqueous soluble P(1,3)-glucan activity.

The present invention also pertains to a novel assay for identifying agents which alter increase or decrease) the effect of underivatized, aqueous soluble P(1,3)-glucan on cellular signal transduction pathways, such as activation of transcriptional regulatory factors. This assay comprises combining underivatized, aqueous soluble P(1,3)-glucan, the receptor for underivatized, aqueous soluble P(1,3)-glucan and an agent to be tested under conditions in which binding of WO 00/13019 PCT/US99/18995 -6underivatized, aqueous soluble P(1,3)-glucan to its receptor occurs conditions suitable for binding of underivatized, aqueous soluble P(1,3)-glucan to the receptor for underivatized, aqueous soluble P(1,3)-glucan). Binding of underivatized, aqueous soluble P(1,3)-glucan to its receptor activates the receptor, which in turn activates a signal transduction as exemplified or measured by a modulation of one or more transcriptional regulatory factors such as those from the NF-KB, NF-IL6 and/or AP-1 families or the enzymes in the respective signal transduction pathways of the transcriptional regulatory pathways. The extent of activation of the selected transcriptional regulatory factor, or pathway enzyme, in the presence of an agent to be tested is determined and compared with the extent of activation of the selected transcriptional regulatory factor, or pathway enzyme, in the absence of the agent to be tested; a difference in the extent of activation indicates that the agent alters the effect of underivatized, aqueous soluble p(1,3)-glucan on activation of the transcriptional regulatory factor or pathway enzyme. An increase in the activation of the transcriptional regulatory factor or pathway enzyme in the presence of the agent indicates that the agent enhances, prolongs or increases, the activation. A decrease in the activation of the transcriptional regulatory factor or pathway enzyme in the presence of the agent indicates that the agent diminishes, shortens or decreases, the activation.

The assays and methods of the present invention can be used to identify agents and drugs for use in treatment of infectious disease, inflammation, autoimmune diseases, ischemia reperfusion injury, cancer, asthma and hypersensitivity disorders. The assays and methods described herein can also be used to identify drugs which prolong or mimic the underivatized, aqueous soluble p(1,3)-glucan effect, and therefore can be used in any therapeutic or prophylactic application in which underivatized, aqueous soluble P(1,3)-glucan can be used, such as for immunomodulation, hematopoiesis, prevention and treatment of infectious disease, platelet production, peripheral blood precursor cell mobilization and wound healing. These agents or drugs act to enhance the effects ofunderivatized, aqueous soluble p(1,3)-glucan by, for example, prolonging the binding of the glucan to its receptor or the effects thereof.

WO 00/13019 PCT/US99/1 8995 -7- The present invention also relates to agents or drugs, such as, but not limited to, peptides or small organic molecules designed with reference to the binding site for underivatized, aqueous soluble p(1,3)-glucan on the receptor for underivatized, aqueous soluble p(1,3)-glucan. In one embodiment, such agents or drugs can be designed to mimic the activity of the receptor binding site in that they bind underivatized, aqueous soluble P(1,3)-glucan, thus decreasing the amount of the P(1,3)-glucan which is available for binding to the receptor and decreasing the activation of downstream events such as signal transduction. The present invention also pertains to an agonist or mimic of underivatized, aqueous soluble P(1,3)-glucan activity with respect to its binding and activation of the receptor for underivatized, aqueous soluble p(1,3)-glucan, such as another conformation of glucan.

Alternatively, the drug or agent can be designed to bind the receptor binding site, rendering it unavailable for binding by underivatized, aqueous soluble P(1,3)glucan; the present invention also relates to antagonists of underivatized, aqueous soluble P(1,3)-glucan binding activity.

The work described herein has application to many areas. For example, it can be used in the monitoring of the underivatized, aqueous soluble P(1,3)-glucan manufacturing process and product characterization for commercial release, to measure P-glucans in fluids, to assess and determine structure-activity relationships of agents that interact with the receptor for the underivatized aqueous soluble P(1,3)glucan. Additionally, this work has application to the targeted delivery of various agents, including drugs and small molecules, to receptor-positive cells such as peripheral polymorphonuclear leukocytes, monocytes, macrophages and epithelial cells. The results described herein can also be used in purification schemes to enrich for both receptor-positive cells and receptor-negative cells, as well as in the generation of anti-receptor antibodies for diagnostic purposes. Furthermore, the methods and assays of the invention allow for specific, highly sensitive detection of p(l,3)-glucans in a test sample, facilitating diagnosis of fungemias and detection of fungal contamination, without requiring expensive equipment or trained personnel.

WO 00/13019 PCT/US99/1 8995 -8- BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graphic representation of dose-dependent binding of 3 H-TH-glucan to NR8383 cells. Solid circles, total binding; open circles, specific binding; solid squares, non-specific binding.

Fig. 2A is a graphic representation of P-glucan selectivity of 3 H-TH-glucan binding to NR8383 cells, determined by competition binding assays using various concentrations of unlabeled competitor low molecular weight P-glucan. Solid circles, TH-glucan; open squares, low molecular weight (LMW)-glucan.

Fig. 2B is a graphic representation of P-glucan selectivity of 3 H-TH-glucan binding to NR8383 cells, determined by competition binding assays using various concentrations of unlabeled competitor very high molecular weight P-glucan. Solid circles, TH-glucan; open squares, very high molecular weight (VHMW)-glucan.

Fig. 2C is a graphic representation of P-glucan selectivity of 3 H-TH-glucan binding to NR8383 cells, determined by competition binding assays using various concentrations of unlabeled competitor P-glucan, scleroglucan. Solid circles, THglucan; open squares, scleroglucan.

Fig. 3A is a graphic representation of p-glucan selectivity of 3 H-TH-glucan binding to lactosylceramide, determined by competition binding assays using various concentrations of unlabeled competitor P-glucan, LMW-glucan. Closed circles, TH-glucan; open squares, LMW-glucan.

Fig. 3B is a graphic representation of P-glucan selectivity of 3 H-TH-glucan binding to lactosylceramide, determined by competition binding assays using various concentrations of unlabeled competitor P-glucan, VHMW-glucan. Closed circles, TH-glucan; open squares, VHMW-glucan.

Fig. 3C is a graphic representation of P-glucan selectivity of 3 H-TH-glucan binding to lactosylceramide, determined by competition binding assays using various concentrations of unlabeled competitor P-glucan, scleroglucan. Closed circles, TH-glucan; open squares, scleroglucan.

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings.

WO 00/13019 PCT/US99/18995 -9- DETAILED DESCRIPTION OF THE INVENTION This invention pertains to the discovery that underivatized, aqueous soluble P(1,3)-glucan (see U.S. Serial No. 07/934,015, filed August 21, 1992, the teachings of which are incorporated herein by reference) specifically binds to a novel receptor located on rat NR8383 macrophages. Underivatized, aqueous soluble P(1,3)-glucans are also described in U.S. Serial Nos. 08/400,488, 08/432,303, 07/934,015, and 08/469,233 and U.S. Patent Nos. 5,322,841, 5,488,040, 5,532,223 and 5,783,569 (the teachings of all of which are incorporated herein by reference). Results of work described herein characterize this receptor for underivatized, aqueous soluble P(1,3)glucan (also known as TH-glucan) and clearly differentiate it from previously described P-glucan receptors, while revealing important information about the mechanism of underivatized, aqueous soluble p(1,3)-glucan biological activity.

The receptor for underivatized, aqueous soluble P(1,3)-glucan is located primarily in rat NR8383 macrophages, particularly alveolar macrophages. As used herein, "receptor" is intended to encompass a traditional receptor molecule as well as a binding site; such a binding site can have an effect of its own or may induce or activate a second molecule or binding site to produce an effect (also known as "unmasking" of a second site; Sandberg et al., Infect. Immun. 63(7):2625-2631 (1995)). As used herein, "receptor" is also intended to include compounds which have an affinity for underivatized, aqueous soluble P(1,3)-glucan; these compounds can be receptor mimics or receptor analogues, and can be isolated from a naturallyoccurring source or can be chemically or synthetically produced. The terms, "NR8383 receptor," "receptor on NR8383 cells," and "receptor from NR8383 cells," refers to a receptor as defined above, which has certain characteristics of the receptor identified on NR8383 cells as described below, such as a proteinaceous composition, an association with a membrane, a certain binding affinity, temperature sensitivity, P-glucan selectivity, trypsin sensitivity, and/or activation of transcription factors.

The receptor (also referred to herein as a "protein receptor" or "proteinaceous receptor") can be present on NR8383 cells, as well as on other types of cells, and can also be isolated separated from cells). For NR8383 cells, see, for example, Helmke, R.J. et al., In Vitro Cell Bevelop. Biol. 23:567-574 (1987); Helmke, R.J. et al., In Vitro Cell Develop. Biol. 25:44-48 (1989).

WO 00/13019 PCTIUS99/1 8995 Binding of labeled, underivatized, aqueous soluble P(1,3)-glucan in the triple helix conformation to the receptor in NR8383 cells was specific, saturable, and fit by a two-site model, with affinities of 45 pM and 170 pM. In addition, binding of underivatized, aqueous soluble P(1,3)-glucan to the receptor on NR8383 cells occurred at either 4°C or 37 0 C. Labeled, underivatized, aqueous soluble P(1,3)glucan (TH-glucan) competed with low molecular weight (LMW)-glucan (approximately 8,000-120,000 daltons) and very high molecular weight (VHMW)glucan (approximately >1,000,000 daltons), as well as for unlabeled, underivatized, aqueous soluble P(1,3)-glucan in the triple helix conformation, for binding to the NR8383 receptor, while scleroglucan did not. Binding of underivatized, aqueous soluble P(1,3)-glucan to the receptor on NR8383 cells was destroyed by pretreatment of cells with trypsin, and recovery of binding activity was dependent upon protein synthesis, indicating that the NR8383 receptor is a protein. In NR8383 cells, LMW-glucan and VHMW-glucan (but not scleroglucan) induced activation of an NF-KB-like factor; underivatized, aqueous soluble p(1,3)-glucan stimulated protein kinase activity of the MAP kinase JNK and the signal some kinase IKKa, but not of the MAP kinases ERK1/2 or p38. These characteristics of the receptor in NR8383 cells differ in binding affinity, temperature sensitivity, P-glucan selectivity, and trypsin sensitivity, from other previously identified underivatized, aqueous soluble P(1,3)-glucan receptors, as described below in the Examples. Thus, the receptor on NR8383 cells represents a previously unidentified receptor for underivatized, aqueous soluble P(1,3)-glucan. The invention therefore pertains to isolated preparations containing the proteinaceous receptor described herein, that binds specifically and selectively), to a ligand that is an underivatized, aqueous soluble P(1,3)-glucan, such as underivatized, aqueous soluble P(1,3)-glucan in a triple helix conformation.

Methods are now available to capture, concentrate or purify P(1,3)-glucans, including yeast or fungal soluble P(1,3)-glucans, yeast or fungal insoluble P(1,3)glucans, as well as to capture, concentrate or purify P(1,3)-glucan-containing organisms, using the receptor from NR8383 cells. In addition, assays to quantify the amount of 1(1,3)-glucan in a test sample are described herein; these assays can be used for diagnosis of fungal infections. Kits useful in the assays and methods are WO 00/13019 PCTIUS99/18995 -11also described. The methods, assays and kits take advantage of the affinity of the P(1,3)-glucan receptor, as described above, for soluble P(1,3)-glucans, such as P(1,3)-glucan and other conformations of glucan LMW-glucan, HMW-glucan and VHMW-glucan).

In accordance with the methods of the invention, a sample known to contain, or thought to contain, fungus or P(1,3)-glucan-containing organisms is obtained.

The test sample can be a liquid or a solid, and can originate from various sources, including complex animal sources mammalian cells or tissues), plant cells or tissues, bacterial cells, viral cells, fungal cells, tissue cultures, environmental sources, food sources, or fermentation process sources. For example, if the test sample is from an animal source, such as from a human individual suspected of having a fungal infection, the test sample can be a biological fluid, such as blood (whole blood, whole blood serum or whole blood plasma), urine, feces, mucus, sputum, bile, ascites fluid, wound secretions, vaginal excretions, synovial fluid, cerebrospinal fluid, peritoneal lavage fluid, lung lavage fluid, ocular fluid, saliva or whole tissue extract; alternatively, the test sample can be solid specimen, such as skin or other tissue. If the individual suspected of having a fungal infection is immunocompromized, the preferred test sample is a urine sample. If the sample is from a plant source, it can be a fluid or a solid specimen, such as plant tissue, plant tissue extract, fruit or fruit extracts, seeds or seed extracts, sap, or plant homogenates. The test sample can also be an environmental source such as water, soil, or soil extracts, or a food source such as prepared foodstuffs, that may be fungally contaminated. Alternatively, the test sample can be a sample from a fermentation process, such as fermentation broth.

If the test sample is high in protein, a de-proteinating step, such as standard trichloroacetic acid or perchloric acid precipitation methods, or precipitation using a standard 70% ethanol precipitation method can be performed prior to performing the assay of the invention (Methods in Enzymology Vol. 182: Guide to Protein Purification (Deutscher, Ed., Academic Press, Inc, San Diego CA, 1990), Chapter 22, pp. 285-306). However, such a step is not necessary to perform the methods of the invention.

WO 00/13019 PCT/US99/18995 -12- In a method of the invention, the proteinaceous receptor as described herein is used to capture or purify p(1,3)-glucans and/or P(1,3)-glucan-containing organisms from a test sample.

In one embodiment of this method of the invention, the receptor can be coupled to a solid phase filter, membrane such as cellulose or nitrocellulose, plastic microtiter plate, dipstick), glass slide), bead latex beads), particle, organic resin, or other organic or non-organic solid phase) or a fluid (e.g.,TRIS buffer or phosphate buffer) phase. Coupling the receptor to the solid or fluid phase can be accomplished by standard methods, including, for example, air drying, heat drying, or chemical reaction. In this embodiment of the method, the test sample is contacted with a receptor coupled to a solid or fluid phase, under conditions that are suitable for binding of any p(1,3)-glucan (or p(1,3)-glucancontaining organisms) that may be present in the test sample to the receptor. P(1,3)glucan or a P(1,3)-glucan-containing organism that is bound to a receptor is referred to herein as a "primary complex". The formation of primary complexes indicates that p(1,3)-glucan, or a P(1,3)-glucan-containing organism, has been "captured" from the test sample. P(1,3)-glucan or a P(1,3)-glucan-containing organism that has been "captured" from the test sample is the p(1,3)-glucan or the P(1,3)-glucancontaining organism that is bound to a receptor.

In another embodiment of this method, the test sample can be attached adsorbed, coated, coupled, covalently attached, attached by affinity binding) to a solid support, such as by dipping the solid support into the test sample, by dropwise application of a fluid test sample with the solid support, or by smearing a solid test sample onto the solid support. If desired, the test sample can be mixed with a liquid, such as saline or any other appropriate biological buffer, prior to attachment to the solid support, such as, for example, in the case of solid test samples. After the test sample is attached to a solid support (forming an "attached test sample"); a receptor is contacted with the attached test sample, under conditions that are suitable for binding of any P(1,3)-glucan that may be present in the attached test sample to the receptor. Receptor that is bound to any P(1,3)-glucan or P(1,3)-glucan-containing organism in the attached test sample is also referred to herein as a "primary complex".

WO 00/13019 PCT/US99/18995 -13- In either embodiment of this method, any primary complexes that have formed can be isolated, in order to obtain, to isolate, to concentrate or to purify the P(1,3)-glucan (or P(1,3)-glucan containing organisms) from the test sample. The primary complexes can be obtained or isolated using standard methods, such as by immunoprecipitation or other means, resulting in concentrated or purified primary complexes, from which concentrated or purified P(1,3)-glucan (or p(1,3)-glucancontaining organisms) can be separated using standard methods (see Current Protocols in Molecular Biology, Vol. II, Ch. 10 (Ausubel, F. et al., eds., John Wiley Sons, 1997).

In another embodiment of the invention, the primary complexes can be detected, in an assay to determine whether P(1,3)-glucan (or P(1,3)-glucancontaining organisms) is present in the test sample. In addition, the amount of primary complexes can be determined: the concentration of the primary complexes correlates positively with the quantity of P(1,3)-glucan (or P(1,3)-containing organisms) present in the test sample. A variety of methods can be used to detect, and/or to determine the quantity of, the P(1,3)-glucan, including detection of the P(1,3)-glucan using an agent that selectively binds the primary complex, such as anti-p-glucan antibodies (monoclonal or polyclonal) or antibody fragments; detection of enzymatic reaction precipitated by P(1,3)-glucan, such as by Limulus amebocyte lysate or Limulus lysate Factor G assay (Mori, T. et al., Eur. J. Clin.

Chem. Biochem 35(7): 553-560 (1997); Hossai, M.A. et al., J. Clin. Lab. Anal.

11:73-77 (1997); Obayashi, T. et al., J Med. Vet. Mycology 30:275-280 (1992), the entire teachings of each of which are incorporated herein by reference); use of enzymes that react with P-glucans; or other means. In a preferred embodiment, the Limulus lysate Factor G assay is used. In another embodiment of the method, the primary complexes can be detected through the use of a detectable label on the receptor, such as a radionucleotide radioummunoassay), dye, fluorescent compound, biotin or streptavidin. If desired, the quantity of primary complexes can be compared to a standard curve generated with a known P(1,3)-glucan standard.

In another embodiment of the invention, a competition assay can be used to determine the quantity of P(1,3)-glucan (or P(1,3)-glucan containing organisms) that is present in the test sample. For example, labeled (for example, with WO 00/13019 PCTIUS99/1 8995 -14radionucleotides, dyes, or fluorescent labels) underivatized, aqueous soluble P(1,3)glucan, or another soluble p(1,3)-glucan, such as low molecular weight (LMW)glucan (approximately 8,000-120,000 daltons, as determined by MALLS), high molecular weight (HMW)-glucan (approximately >210,000 <1,000,000, as determined by MALLS) or very high molecular weight (VHMW)-glucan (approximately >1,000,000 daltons, as determined by MALLS) is incubated with the proteinaceous receptor described herein and the test sample, under conditions suitable for binding of underivatized, aqueous soluble P(1,3)-glucan to its binding agent. Subsequently, the amount of primary complexes containing labeled, underivatized, aqueous soluble p(1,3)-glucan is determined. This amount is compared with the amount of primary complexes generated by incubation of labelled underivatized, aqueous soluble p(1,3)-glucan with the receptor in a control sample a sample having a known amount of unlabeled P(1,3)-glucan) or a series of control samples a standard curve). The amount of 1(1,3)-glucan in the test sample is inversely proportional to the amount of primary complexes containing labelled, underivatized, aqueous soluble P(1,3)-glucan.

Detection and quantitation of the presence of 1(1,3)-glucan, or of 1(1,3)glucan containing organisms, is useful in the diagnosis of fungemias or fungal contamination. To diagnose fungal infection in an individual, for example, a sample, such as a biological fluid sample, is obtained from the individual. The sample is assayed for the presence of P(1,3)-glucan, as described in detail above.

The presence of 1(1,3)-glucan in the test sample is indicative of the presence of a component of fungal cell walls, and is therefore indicative of fungal presence, contamination, or infections. Similarly, the presence of 1(1,3)-glucan in a sample from an environmental source, or a food source, is indicative of fungal presence or contamination. In addition, quantitation of the amount of 1(1,3)-glucan, or of P(1,3)-glucan containing organisms, can provide information regarding the severity of the infection or contamination. The methods and assays of the invention provide accurate and specific identification of the presence of 1(1,3)-glucans in a test sample, thereby facilitating diagnosis and treatment of fungal infection and contamination.

WO 00/13019 PCT/US99/18995 In addition, as a result of the work described herein, it is possible to monitor the underivatized, aqueous soluble P(1,3)-glucan manufacturing process and characterize the product. That is, test samples can be included in a standard competition receptor binding assay to allow characterization of the molecule in terms of relative affinity for the receptor. This characterization will yield information with respect to some or all of the following characteristics: batch to batch quality, identification of product as a P(1,3)-glucan of a particular conformation and purity of the product.

Test samples can also be included in standard capture or competition assays and compared with a standard sample to elucidate structure-activity relationships.

Alternatively, the test sample binding can be tested directly after radiolabeling.

Samples can also be tested in an underivatized, aqueous soluble P(1,3)-glucan receptor-mediated assay to test for inhibition or stimulation of these functions. For example, such assays can be used in the development of polymer or small molecule receptor agonists or to develop a receptor antagonist to inhibit an inappropriate immunoenhancement, such as to prevent a transplant rejection which might occur as a result of an opportunistic fungal infection during immunosuppressive therapy.

The discoveries disclosed herein can also be used to target delivery of various agents to receptor-positive cells. For example, various agents can be conjugated chemically conjugated, cross-linked or covalently bonded) to underivatized, aqueous soluble p(1,3)-glucan to produce a conjugate molecule which can be targeted to receptor-positive cells such as macrophages. Such a targeted delivery system can be used to enhance the delivery of agents such as antimicrobials for resistant intracellular pathogens mycobacterium, leishmania, malaria), cytotoxins for receptor-positive leukemias, genes for gene therapy for enhanced cytokine production or replacement for dysfunctional enzymes), or antigens for enhanced presentation and production of specific antibodies or T-cell activation.

The binding specificity of the underivatized, aqueous soluble P(1,3)-glucan to its receptor provides a method for purification of both receptor-positive and receptor-negative cells cells which do not contain the receptor); for example, underivatized, aqueous soluble P(1,3)-glucan can be affixed to a solid matrix and WO 00/13019 PCT/US99/18995 -16used as an affinity matrix to positively select receptor-positive cells or to negatively select receptor-negative cells. Similarly, anti-receptor antibodies can be used in place of the immobilized underivatized, aqueous soluble P(1,3)-glucan. Cells which are purified by this method can subsequently be expanded for use in cell therapy.

Furthermore, the present invention makes possible the generation of antireceptor antibodies for diagnostic purposes. Monoclonal or polyclonal antibodies can be produced using enriched or purified receptor preparations by standard techniques. Thus, the present invention also relates to antibodies which bind the receptor for underivatized, aqueous soluble P(1,3)-glucan. For instance, polyclonal and monoclonal antibodies which bind to the described receptor are within the scope of the invention. A mammal, such as a mouse, hamster, goat or rabbit, can be immunized with an immunogenic form of the receptor an antigenic portion of the receptor which is capable of eliciting an antibody response). Techniques for conferring immunogenicity include, for example, conjugation to carriers or other techniques well known in the art. The antigen can be administered in the presence of an adjuvant, and the progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibody. Antibodies to the proteinaceous receptor described herein can be produced by standard techniques such as, for example, those described in Current Protocols in Immunology (John Wiley Sons, 1997). Such antibodies can be used to identify alterations in receptor-positive or receptor-negative cell populations which reflect disease pathology response to cryptic fungal infection or leukemia).

This invention also pertains to a method of altering activating or deactivating) signal transduction pathways, for example through modulation of transcriptional regulatory factors or signal transduction enzymes (pathway enzymes) in receptor-positive cells, cells which contain the receptor for underivatized, aqueous soluble P(1,3)-glucan described herein. In one embodiment of the invention, the transcriptional regulatory factor is from the NF-KB and/or NF-IL6 and/or AP-1 families of transcriptional regulatory factors. For example, the transcription factor can be NF-KB, NF-IL6 or AP-1.

WO 00/13019 PCTIUS99/18995 -17- According to the method of the present invention, the activity of the receptor for underivatized, aqueous soluble p(1,3)-glucan is activated through binding of an underivatized, aqueous soluble P(1,3)-glucan, whereby a signal transduction pathway consisting of a cascade of enzymes) is activated and thereby regulated by a transcriptional regulatory factor from the NF-KB, NF-IL6 and/or AP-1 families) is activated. 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 activated by an agent which mimics the binding and activation ability of an underivatized, aqueous soluble p(1,3)-glucan another conformation of glucan). In a particular embodiment, the transcriptional regulatory factor or pathway enzyme is activated as a result of ligand binding. In another embodiment, the activity of the transcriptional regulatory factor or pathway enzyme is decreased, either partially or totally, by the binding of an agent which binds the receptor (and thus excludes the underivatized, aqueous soluble P(1,3)-glucan), but lacks the ability to activate the receptor.

Other signal transduction pathways which can be altered by the methods of the present invention include the ras/raf-1/MAP kinase (ERK) pathway, the Gprotein/phospholipase C/protein kinase C pathway, the non-G-protein/phospholipase C/protein kinase C pathway, the JAK/STAT pathway, the phospholipase A pathway, G-protein/phospholipase D/phosphatidic acid pathway, the c-AMP-dependent pathway, the c-Jun N-terminal kinase (JNK, also known as stress-activated protein kinase (SAPK)) pathway, the IKB kinase a (IKKa) pathway, the protein tyrosine kinase pathway, and the oxygen radical pathway. In each pathway, an appropriate activator or indicator of the signal pathway is activated by binding of underivatized, aqueous soluble P(1,3)-glucan to its receptor, and modulation of this binding can alter the corresponding signal transduction event.

The present invention also pertains to a novel assay for identifying agents which alter the effect of underivatized, aqueous soluble p(1,3)-glucan on signal transduction pathways, such as activation of transcriptional regulatory factors or pathway enzymes. This assay comprises combining underivatized, aqueous soluble p(1,3)-glucan, the receptor for underivatized, aqueous soluble P(1,3)-glucan and an WO 00/13019 PCT/US99/18995 -18agent to be tested under conditions in which binding of underivatized, aqueous soluble P(1,3)-glucan to its receptor occurs conditions suitable for binding of underivatized, aqueous soluble P(1,3)-glucan to the receptor for underivatized, aqueous soluble P(1,3)-glucan). Binding ofunderivatized, aqueous soluble P(1,3)glucan to its receptor activates the receptor, which in turn activates a signal transduction pathway as shown by transcriptional regulatory factors such as those of the NF-KB and/or NF-IL6 and/or AP-1 families. The extent of activation of the selected transcriptional regulatory factor or pathway enzyme in the presence of an agent to be tested is determined using radiolabeled DNA oligonucleotides specific for the transcriptional regulatory factor, as in the Examples) and compared with the extent of activation of the selected transcriptional regulatory factor or pathway enzyme in the absence of the agent to be tested; a difference in the extent of activation indicates that the agent alters the effect ofunderivatized, aqueous soluble p(l,3)-glucan on activation of the transcriptional regulatory factor or pathway enzyme. An increase in the activation of the transcriptional regulatory factor or pathway enzyme in the presence of the agent indicates that the agent enhances, i.e., prolongs or increases, the activation. A decrease in the activation of the transcriptional regulatory factor or pathway enzyme in the presence of the agent indicates that the agent diminishes, shortens or decreases, the activation.

Kits useful in the methods and assays described above are also contemplated in the invention. Such a kit comprises primarily the components and reagents described for the methods and assays, such as a solid phase, detectable labels, P(1,3)-glucan binding agent, and/or labeled, underivatized, aqueous soluble, P(1,3)glucan. In one embodiment, the kit comprises the receptor described herein as a p(1,3)-glucan binding agent. In a preferred embodiment, the p(1,3)-glucan binding agent is attached to a solid support, such as a filter, a membrane such as cellulose or nitrocellulose, a plastic support microtiter plate, dipstick), a glass support slide), beads latex beads), particles, an organic resin, or other organic or nonorganic solid phase.

The following Examples are offered for the purpose of illustrating the present invention and are not to be construed to limit the scope of this invention.

WO 00/13019 PCT/S99/1 8995 -19- The teachings of all references cited herein are hereby incorporated herein by reference.

EXAMPLE 1 Materials and Methods A. Materials Ham's F-12 tissue culture medium was purchased form Life Technologies (Grand Island, NY); fetal calf serum (fcs) and lipopolysaccharide (LPS, serotype 0128:B12) were purchased from Sigma (St. Louis, MO); TH-glucan, low molecular weight-glucan (LMW-glucan, approximately 8,000-120,000 daltons), high molecular weight-glucan (HMW-glucan, approximately >210,000 <1,000,000 daltons), very high molecular weight-glucan (VHMW-glucan, approximately >1,000,000 daltons) were prepared at Alpha-Beta Technology, Inc. according to methods described in U.S. Patent Nos. 5,622,939, 5,783,569 and 5,817,643; Scleroglucan (ACTIGUMT

M

was obtained from Sanofi Bio-Industries (Paris, France). Scleroglucan used for assays in this report was modified by mild formic acid hydrolysis, neutralization and size fraction on gel permeation chromotography to achieve the equivalent hydrodynamic volume as TH-glucan. All P-glucan samples tested negative for endotoxin contamination by Limulus amoebocyte lysate assay. All other reagent grade materials were purchased from either Sigma or Boehringer Mannheim (Indianapolis, IN). GST fusion proteins ATF-2, ELK-1, c-jun were obtained from New England Biolabs (Beverly, MA); anti-ERK1/2, -p38, -JNK, and -IKKa antibodies, as well as GST fusion protein IicBa, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For NR8383 cells, see, for example, Helmke, R.J. et al., In Vitro Cell Develop. Biol. 23:567-574 (1987); Helmke, R.J. et al., In Vitro Cell Develop. Biol. 25:44-48 (1989). Molecular weights were determined by MALLS.

B. Preparation of 3 H-TH-glucan TH-glucan (Alpha-Beta Technology, Worcester, MA) was incubated with NaIO 4 (10 mg/ml; Sigma) in sterile pyrogen-free (SPF) water for 72 hours at room temperature. The periodate was quenched by the addition of 0.5 ml ethylene glycol.

The oxidized TH-glucan was dialyzed against SPF water, and then reductively WO 00/13019 PCT/US99/1 8995 labeled with 100 mCi of NaB 3

H

4 (New England Nuclear, Boston, MA). 3

H-TH-

glucan was separated from tritiated low molecular weight degradation products by dialysis and ultrafiltration. Purity of the labeled product was assessed by gel permeation chromatography.

C. NR8383 whole cell binding assay NR8383 cells were grown in Ham's F-12 medium containing 15% fetal bovine serum (F-12/fcs) using standard tissue culture techniques. Cells were harvested at a cell density of approximately 3x10 5 cells/ml by scraping and centrifugation (400 x g, minutes, room temperature). Cells were resuspended in PBS and combined with 3 H-TH-glucan (1 plg/ml final) and either saline or competitor as indicated in the figures. Cell concentration was 0.25-1.0 x10 6 cells/well/200 ul. The binding reaction was allowed to proceed for 60 minutes at 37 0 C. At the end of the incubation period, cells were washed 2 x 250 ul PBS by centrifugation, solubilized with 0.1 N NaOH, and radioactivity quantitated by liquid scintillation counting.

The dose-dependence of 3 H-TH-glucan binding to NR8383 cell was assessed by incubating 3.3 x 10 6 NR8383 cells/ml with increasing concentrations (as indicated in Fig. 1) of 3 H-TH-glucan with (non-specific binding) or without (specific binding) 1000-fold excess unlabeled TH-glucan in 350 tl Hank's buffered saline (Ca*/Mg free; HBSS-) for 60 minutes at 4°C. Cells were rinsed twice in HBSS- by centrifugation, cell pellets solubilized, and radioactivity quantitated by liquid scintillation counting. Preliminary experiments indicated that equilibrium binding levels were reached by 60 minutes. The 4°C incubation temperature in these experiments was used to inhibit the process of ligand/receptor internalization in order to facilitate analysis of the equilibrium binding data. Samples of the incubation medium were saved at the end of the incubation for determination of free 3 H-TH-glucan. Specific binding was calculated as the difference between total binding and non-specific binding. Data was fit to a two site/two affinity model by nonlinear regression analysis (Kaleidagraph, Synergy, Reading, PA) according to the equation B, E {Bm Bma[L/(L+Kd 2 where B,represents the total amount of labeled ligand bound, L represents the concentration of labeled ligand, WO 00/13019 PCT/US99/18995 -21- Bman represents maximal binding capacity of site n, and Kdn is the apparent dissociation constant of site n.

D. Trypsinization ofNR8383 cells Cells were harvested as described above and resuspended in sterile PBS containing 5 mM EDTA. Cells were aliquoted into separate tubes, re-centrifuged, and then resuspended into 1 ml PBS/EDTA with or without 0.25% trypsin Cells were incubated at 35 0 C for 45 minutes. At the end of the incubation period, ml soybean trypsin inhibitor w/v) was added, followed by 10 ml F- 12/fcs. Cells were centrifuged as above and resuspended in either 0.7 ml PBS for immediate assay of binding activity, or in 10 ml F-12/fcs with or without cycloheximide (1 jg/ml) and cultured for an additional 24 hours. For assay of binding activity, cells were counted in a hemocytometer and cell numbers equalized.

Cells were then assayed as described above except that the binding reaction was done at 4°C in the presence or absence of 100 ug/ml unlabeled TH-glucan. Cells were processed and radioactivity quantitated as described.

E. Binding to Lactosylceramide 3 H-TH-glucan binding to lactosylceramide was measured as previously described Patent application number 08/990,125, attorney docket no. ABY97- 04, filed on December 12, 1997; the entire teachings of which are incorporated herein by reference). Briefly, lactosylceramide (Sigma Chemical Co.) was dissolved in ethanol at 1 mg/ml. Aliquots (20 pl) were applied in triplicate to the wells of a 96-well polystyrene plate (Costar) and air dried. Plates were blocked by incubation with 300 pl of 1% gelatin in PBS at 37 0 C for 1-2 hours. Plates were then rinsed with PBS (2 x 200pl). Fifty microliters of either PBS or unlabeled polysaccharide was added to each well, plates were equilibrated at 37 0 C, then 3

H-

TH-glucan added (1 pg/ml, 100 pl final). Plates were incubated for 1-2 hour at 37 0 C, then rinsed twice with 200pl PBS. Radioactivity was solubilized with 150 pl SolvableTM (DuPont NEN Wilmington, DE) and counted.

WO 00/13019 PCT/US99/1 8995 -22- F. Immunoprecipitation/kinase assays NR8383 cells were incubated in F-12 0.1% fcs overnight at 5x10 6 cells/ml x2 ml in 6-well plates. Cells were then incubated with either LPS (1 pg/ml) or THglucan (3 pg/ml) for the indicated times at 37 0 C. Cells were placed on ice, scraped from the wells and added to 10 ml ice cold PBS. Cells were centifuged at 400 x g for 5 minutes at 4°C, then resuspended in 1 ml PBS at 4 0 C. Cells were centrifuged once more and then resuspended in lysis buffer (20 mM Tris pH 7.4, 137 mM NaC1, 2 mM EDTA, 25 mM b-glycerophosphate, 2 mM Na pyrophosphate, 10% glycerol, 1% Triton X-100) containing 1 pg/ml leupeptin, aprotinin, pepstatin, 1 mM NaVO 4 1 mM PMSF, incubated for 10 minutes at 4 0 C, then centrifuged at 15,000 x g for minutes. Protein content was determined by the Bradford assay (Pierce). Equal protein loads (100-500 gg) were added to protein A sepharose beads (Pierce) prebound with kinase-specific antibody (see figures) as described below. The immunoprecipitation reaction mixture was incubated 2-4 hours with rotation at 4 0

C.

Beads were then washed 3 x 1 ml lysis buffer and 1 x 1 ml with kinase reaction buffer (25 mM HEPES pH 7.4, 25 mM b-glycerophosphate, 25 mM MgCl 2 2 mM NaVO 4 and finally resuspended in 50 pl kinase reaction buffer containing 2 mM DTT. The kinase reaction was then initiated by combining 20 pl beads, 22 p.

1 reaction mix (20 pl kinase reaction buffer, 1 pl 1 mM ATP, 1 pl/10 pCi g- 32

P-ATP),

0.5 pl of 2 pg/ml kinase substrate. Reaction proceeded for 15-20 minutes at room temperature and the reaction stopped by addition of 10 pl 4x Laemmli buffer and boiling for 5 minutes. Samples were centrifuged and an aliquot of the reaction mixture run on a 10% SDS-PAGE followed by autoradiography.

Kinase-specific antibodies were pre-bound to protein A sepharose beads as follows: beads were washed twice in 1 ml lysis buffer, centrifuged (15,000 x g for seconds), and resuspended in 0.5 volumes lysis buffer. Twenty microliters of bead suspention were aliquoted into microfuge tubes, and 2 pl antibody (approximately 0.4 pg) were added. The bead/antibody mixture was allowed to incubate for 20-30 minutes on ice before addition of cell lysates.

WO 00/13019 PCT/US99/18995 -23- G. Electrophoretic mobility shift assays (EMSA) EMSA were performed essentially as described in Adams, et al., J. Leuk. Biol.

62 865-873 (1997). In brief: Cell Stimulations LPS (1 pg/ml), TH-glucan (3 gg/ml), low molecular weight-glucan (LMWglucan; 3 or 30 ptg/ml), high molecular weight-glucan (VHMW-glucan; 3 pg/ml), or scleroglucan (3 jig/ml) were added to the medium and 37 0 C incubations continued for 0.5-1.0 hr. After stimulation cells were scraped into ice cold PBS, rinsed once with PBS, then used for nuclear extractions.

PMN were prepared from donor blood anti-coagulated with acid citrate dextrose. Following sedimentation in 1.5% dextran cells were centrifuged (400 x g, 7 minutes) and resuspended in autologous plasma. Cells were then layered over a Ficoll gradient (Lymphocyte Separation Medium; Organon Teknika, Durham, NC) and centrifuged (400 x g, 30 minutes). Cells recovered from the pellet were hypotonically lysed to remove residual RBC. The remaining cells were neutrophils as judged by morphological criteria, and were then stimulated as described above prior to preparation of nuclear extracts.

Nuclear Extractions Nuclear extracts for EMSA were prepared as described previously (Adams, et al., 1997)) using 1.0-2.0 x 10 7 cells per sample. All buffers were freshly supplemented with DTT (0.5 mM), protease inhibitor cocktail, and phosphatase inhibitor cocktail. Protein concentrations were determined by Bradford assay (Pierce) against a BSA standard.

Electrophoretic Mobility Shift Assays (EMSA) Transcription factor activations were assayed using an EMSA as described previously (Adams, et al., (1997)). NF-KB consensus synthetic duplex probe was as described (Adams, et al., (1997); see also Lenardo, M.J. and Baltimore, Cell 58:227-229 (1989)). 32 P-labeled duplex probes were prepared with polynucleotide kinase. Labeled probe (0.5 pmol) was mixed with 3 lg of nuclear extract protein in a solution containing 10 mM Tris-HCl pH 7.5, 50 mM NaC1, 1 mM EDTA, 1 mM WO 00/13019 PCT/US99/18995 -24- DTT, 5% glycerol, 0.02% P-mercaptoethanol, 0.1-1.0 gg of poly(dl/dC) (Pharmacia). Reactions were incubated at 25 C for 20 min then electrophoresed under non-denaturing conditions through 4% polyacrylamide gels in 0.5X TBE buffer. Bands were visualized by autoradiography.

EXAMPLE 2 3 H-TH-glucan binding to intact NR8383 cells A. Dose-dependent binding of H-TH-glucan to NR8383 cells NR8383 cells were incubated with 3 H-TH-glucan at increasing concentrations in the presence (non-specific) or absence (total) of 1000-fold excess unlabeled THglucan for 60 minutes at 4 0 C. At the end of the incubation, unbound radioactivity was removed by washing, and the amount of cell-associated radioactivity determined as described above. Specific binding was determined by subtracting non-specific from total radioactivity. The results are shown in Fig. 1; the specific binding curve was fit by a nonlinear regression analysis of a two-site model as described above. The data in Fig. 1 demonstrate that 3 H-TH-glucan binding to the NR8383 cells was dose-dependent, specific, and saturable. NR8383 cells bound increasing amounts of 3 H-TH-glucan as the concentration of ligand was increased (total). Addition of unlabeled TH-glucan to the incubation mixture inhibited the amount of 3 H-TH-glucan associated with the cells (non-specific). After subtraction of the non-specific bound from the total bound, the specific binding was found to be saturable at about 30 fin/10 6 cells. Non-linear regression analysis of the specific binding curve fit a two-site binding model, with apparent dissociation constants Kdl and Kd2 of 45 pM and 170 pM, respectively. The number of binding sites was 3,200/cell and 18,000/cell for the high and low affinity sites, respectively. The binding activity observed at 4 0 C for NR8383 cells contrasts with that observed for lactosylceramide, which shows no binding activity at 4°C (data not shown).

B. Selectivity of 3 H-TH-glucan binding to NR8383 cells and lactosylceramide The selectivity of P-glucan binding to the 3 H-TH-glucan binding site on NR8383 cells was determined by competition binding assays. In these assays, WO 00/13019 PCT/US99/18995 NR8383 cells were co-incubated with 3 H-TH-glucan and various concentrations of unlabeled competitor P-glucan (LMW-glucan, VHMW-glucan; or scleroglucan) for minutes at 37 0 C. Unbound radioactivity was removed and cell-associated radioactivity determined as described above. The results of this type of experiment are shown in Fig. 2A-C (Fig. 2A: LMW-glucan; Fig. 2B: VHMW-glucan; Fig. 2C: scleroglucan). Data points represent the mean control binding coefficient of variation; TH-glucan was included as a positive control in each experiment.

Unlabeled TH-glucan, LMW-glucan, and VHMW-glucan were able to compete for 3 H-TH-glucan binding, whereas scleroglucan was not an effective competitor in the concentration range tested.

Lactosylceramide has been identified as the TH-glucan receptor on human neutrophils Patent application number 08/990,125, attorney docket no.

ABY97-04, filed on December 12, 1997; the entire teachings of which are incorporated herein by reference). The ability of these same P-glucans to compete for binding of 3 H-TH-glucan to lactosylceramide was therefore investigated.

Lactosylceramide was coated onto 96-well plates as described above, and incubated with 3 H-TH-glucan (1 gg/ml) in the presence of various concentrations of unlabeled P-glucans (LMW-glucan; VHMW-glucan; scleroglucan) for 60-90 minutes at 37 0

C.

Wells were rinsed and the remaining radioactivity was measured as described above.

The results are shown in Fig. 3A-C (Fig. 3A: LMW-glucan; Fig. 3B:VHMWglucan; Fig. 3C: scleroglucan). Unlabeled TH-glucan and VHMW-glucan were effective competitors of 3 H-TH-glucan binding to lactosylceramide. In contrast to the results obtained with NR8383 cells, LMW-glucan was unable to compete for binding, while scleroglucan was found to be an effective competitor. This data clearly indicates that the NR8383 receptor has different P-glucan selectivity than lactosylceramide.

C. Trypsin sensitivity ofNR8383 cell 3 H-TH-glucan binding activity The 3 H-TH-glucan receptor on NR8383 cells was found to be sensitive to trypsinization. Experiments were conducted in which NR8383 cells were incubated with trypsin prior to measurement of 3 H-TH-glucan binding activity. Table 1 shows the results from the experiments. Cell numbers were normalized for the binding WO 00/13019 PCT[S99/18995 -26assay, and specific binding (total binding nonspecific binding) was normalized to respective control values at the 0 and 24 hour time points. Trypsin pre-treatment eliminated 96% of specific binding relative to untreated control cells. When cells were incubated in normal medium for 24 hours after trypsinization, binding activity recovered to almost 50% beyond control levels. This recovery was inhibited by the protein synthesis inhibitor cycloheximide (1 ag/ml). Interestingly, when cycloheximide was added to untreated control cells, binding activity was reduced by 94%. This suggests that a labile protein is necessary for continued expression of H- TH-glucan binding activity. This could represent the receptor per se, or a protein which stabilizes either the protein or its mRNA. Nevertheless, these results strongly indicate that the NR8383 receptor is a protein, and not a glycosphingolipid.

Table 1: Effect of Trypsin and Cycloheximide on 3 H-TH-glucan Binding to NR8383 Cells Treatment Percent control binding, Percent control binding, 0hr 24 hr Control 100 100 Control cycloheximide 6 Trypsin 4 148 Trypsin cycloheximide EXAMPLE 3 Signal Transduction events initiated by TH-glucan in NR8383 cells A. Activation of an NF-kB-like transcription factor TH-glucan-induced signal transduction through lactosylceramide on human PMN and murine BMC2.3 macrophages has been shown to activate an NF-rB-like nuclear transcription factor Patent application number 08/990,125, attorney docket no. ABY97-04, filed on December 12, 1997; the entire teachings of which are incorporated herein by reference). The ability of TH-glucan, LMWglucan,VHMW-glucan, and scleroglucan to activate NF-KB-like DNA binding WO 00/13019 PCT/US99/18995 -27activity in NR8383 cells and human PMN was compared using Electrophoretic Mobility Shift Assays (EMSA). Cells were incubated with the P-glucans (control; LPS, 1 plg/ml; TH-glucan, 3 jig/ml; scleroglucan, 3 jig/ml; LMW-glucan, 3 ug/ml or gg/ml; VHMW-glucan, 3 ljg/ml). Nuclear extracts were prepared for determination of NF-KB activation by EMSA as described above. In NR8383 cells, TH-glucan, VHMW-glucan, and LMW-glucan induce NF-KB-like DNA binding activity, but scleroglucan does not (data not shown). In PMN, TH-glucan and scleroglucan induce the NF-kB-like DNA binding activity, while LMW-glucan does not (data not shown: VHMW-glucan was not tested in these cells). Thus, signal transduction initiated by these various P-glucans in rat NR8383 cells and human PMN correlates with their ability to compete for 3 H-TH-glucan binding.

B. Activation of Protein Kinases Finally, the early signal transduction events induced by TH-glucan in NR8383 cells were investigated. In particular, immunoprecipitation coupled to protein kinase assays was used to determine which protein kinase cascades are involved in THglucan signaling. NR8383 cells were incubated with LPS (1 Ig/ml, positive control), TH-glucan (3 ug/ml), or no addition for 30 minutes at 37 0 C. Cells were lysed, immunoprecipitated with kinase-specific antibody, and a kinase assay performed as described above. Equal amounts of protein were used for immunoprecipitations within each experiment. TH-glucan does not activate the MAP kinases extracellular mitogen-regulated kinase (ERK1/2) (kinase substrate was ATF-2-GST); or p38 (kinase substrate with ELK-1-GST) (data not shown). In contrast, TH-glucan rapidly and transiently activates the MAP kinase c-Jun Nterminal kinase (JNK, also known as stress-activated protein kinase or SAPK; kinase substrate was c-jun-GST) (data not shown). TH-glucan also rapidly and transiently activates the IKB kinase alpha (IKKa; kinase substrate was IiBa-GST), the kinase that phosphorylates IBa, leading to activation of NF-rB-like transcription factors (data not shown).

WO 00/13019 PCT/US99/1 8995 -28- EXAMPLE 4 Comparison of TH-glucan and its Conformers in an Infection Model A. Materials and Methods TH-glucan and Conformers TH-glucan (Alpha-Beta Technology) and certain conformers were used in studies of their effect on an infection model. Table 2 illustrates the molecular weights of TH-glucan and the conformers used in this study. Because the molecular weight of conformers can change with the temperature in the GPC column, conformers separated at room temperature (25 C) and at a higher temperature (37 0 C) were tested.

Table 2 Molecular Weights of TH-glucan and Conformers Conformer Average Molecular Weight (daltons) VHMW (very high molecular weight) >1,000,000 HTH (high triple helix) 25 C 244,100 HTH 37 0 C 195,200 TH (triple helix) glucan 150,000 LTH (low triple helix) 25 0 C 104,300 LTH 37 0 C 80,480 LMW (low molecular weight) 11,000 The average molecular weights were measured by a gel permeation chromatography/multi-angle laser light scattering (GPC/MALLS). HTH and LTH were prepared by fractionating samples of TH-glucan.

Animals Virus-antibody free Wistar rats were purchased from Charles River Breeding Laboratories (Wilmington, MA). Animals were housed according to the National Institutes of Health guidelines and were provided food and water ad libitum. Rats were quarantined for 7 days prior to being entered into experiments. Body weights of rats ranged from 160 g to 210 g at the time of experimentation.

WO 00/13019 PCT/US99/1 8995 -29- Drug Administration TH-glucan and conformers, as well as whole glucan particles (WGP), were dosed intramuscularly (IM) using a 25-gauge needle at 1 mg/kg at 48 h, 24 h and 4 h before bacterial challenge and at 4 h after bacterial challenge. In addition, as a positive control, WGP was also given intraperitoneally (IP) at a dose of 5 mg/kg at 24 h and 4 h before bacterial challenge. WGP was made as a suspension with sterile saline and was vigorously vortexed before each injection.

Bacteria A multiple antibiotic resistant clinical isolate of S. aureus was chosen for use in these studies. Onderdonk, et al., Infect. Immun. 60: 1642-1647 (1992).

Bacteria were expanded in Nutrient Broth (NB; Bifco Laboratories, Detroit, MI) for 8 h at 37 0 C. Sterile glycerol was added at a final concentration of 20% and stock aliquots frozen at -80 0 C until used. To determine the viability of stock bacterial, a frozen stock sample was thawed, serially diluted, ant 40 tl samples plated onto Nutrient Agar (NA; Difco Laboratories, Detroit, MI) plates. Plates were cultured at 37 0 C for 24 h, the bacterial colonies were counted, and the number of bacterial colony forming units (CFU) per ml of stock was calculated. To prepare bacterial inoculum for in vivo studies, stock cultures were diluted to the desired number of CFU/ml in Phosphate Buffered Saline (PBS; Gibco Life Technologies, Grand Island, NY) containing 1% dextran sulfate and a final concentration of 5% barium sulfate (wt/vol). A 0.5 ml aliquot of the appropriately diluted bacteria was aseptically placed into 2 cm long x 0.5 cm diameter gelatin capsules (Eli-Lilly Inc., Indianapolis, IN) and the capsules were implanted into the peritoneal cavity of rats as described below.

Surgery Rats were anesthetized by im injection of a mixed anesthetic cocktail consisting of Ketamine (Fort Dodge Laboratores Inc., Fort Dodge, IA), PromAce (Ayerst Laboratores Inc., Rouses Point, MY), Xylazine (Phoenix Scientific Inc., St.

Joseph, MO) and saline (750 mg, 10 mg, 100 mg and saline to 20 ml) using a gauge needle. The anesthesia was adjusted for each rat based on body weight by WO 00/13019 PCTIUS99/18995 administering 0.0019 mlg body weight. After administering the anesthesia, the abdomen of each rat was shaved, cleaned with iodine solution, and a 1.5 cm anterior midline incision was made through the abdominal wall and the peritoneum. The gelatin capsule containing 10 8 S. aureus CFU was immediately placed into the peritoneal cavity and the incision was closed with interrupted 3-0 silk sutures. The total duration of surgery was less than 2 minutes. Following surgery, animals were housed five per cage and monitored every 15 min for the first 2 h, every 30 min for the next h, and then three times per day for the duration of the experiment.

Blood Sampling and Analysis Animals were anesthetized with O 2

:CO

2 and 2 ml of blood was obtained by cardiac puncture using a 3 ml syringe with 20 gauge needle. Immediately after blood was collected, approximately 1.5 ml was expelled into a 1.7 ml Eppendofr centrifuge tube containing 5 units of heparin (Elkins-Sinn Inc., Cherry Hill, NJ).

Each animal was then humanely euthanized with CO 2 To obtain accurately quantifiable blood CFU levels, two concentrations of blood from each animal, 1:10 dilution and undiluted blood were cultured. Twenty ml of 50 0 C Tryptic Soy Agar (Becton Dickinson Microbiology Systems, Cockeysville, MD) was plated into a sterile petri plate and a 0.5 ml aliquot of diluted blood or undiluted blood immediately added to the plate and thoroughly mixed into the agar by swirling the plate. Once the agar solidified, plates were incubated at 37 0 C for 48 h, and then the plate contained 20-200 colonies were selected for the colony enumeration. Blood CFU data are expressed as log CFU/ml of blood. The remaining blood from each sample was used for blood cell counts. Total white blood cell 9WBC), red blood cell (RBC), and platelet (PLT) counts were performed on a System 9010+ Hematology Analyzer (Biochem Immunosystems Inc., Allentown, PA).

Statistical Analysis Results are expressed at the mean standard error of the mean (SEM) of data obtained from replicate experiments (2 -6 experiments, containing 20-80 animals).

Unpaired t-tests (treatment group vs. saline) were performed using EXCEL WO 00/13019 PCr/US99/1 8995 -31software (Microsoft Corporation, Redmond, WA) and differences were considered significant at p 0.05.

B. Results Effects of TH-glucan and Conformers on Blood CFU Levels Rats were administered TH-glucan, a conformer, or WGP and blood CFU levels were determined at 48 h after challenge with S. aureus. Results are shown in Table 3.

Table 3 Effect of TH-glucan and Conformers on Blood CFU Levels Treatment Log CFU/ml blood p value Saline 2.09 0.16 WGP (IP) 0.47 ±0.15 <0.001 WGP (IM) 1.09 ±0.45 <0.01 VHMW (very high molecular weight) 1.55 ±0.15 <0.05 HTH (high triple helix) 25 C 1.70 ±0.19 <0.05 HTH 37 0 C 1.63 ±0.18 <0.05 TH-glucan 1.62 ±0.17 <0.05 LTH (low triple helix) 25 C 1.31 ±0.15 <0.05 LTH 37 0 C 1.59 ±0.22 <0.05 LMW (low molecular weight) 1.62 ±0.12 <0.05 As can be seen, rats treated with TH-glucan, as well as all other materials tested in these studies, exhibited significantly lower blood CFU levels than saline-treated rats at 48 h after bacterial challenge (P 0.05). The greatest reduction in blood CFU levels were observed in the rats treated with insoluble WGP IP and IM (P 0.001 and p 0.01).

Effect of TH-glucan and Conformers on Peripheral Blood Cell Counts Blood cell counts were performed at 48 h following S. aureus challenge. Total WBC counts were elevated in rats treated with TH-glucan and all conformers, as WO 00/13019 PCTIUS99/1 8995 -32well as WGP, although statistical significance could only be seen in the rats treated with TH-glucan, HTH 25 C, LTH 25 C and LTH 37 C. Although TH-glucan had no effect on PLT counts, rats treated with VHMW, HTH25 C, or HTH 37 0 C conformers with molecular weights greater than 190,000), or WGP (IM) or WGP (IP) exhibited significantly increased peripheral PLT counts. RBC values did not change except for slight elevations in rats treated with HTH 25 C, LMW and WGP These results are summarized in Table 4.

Table 4 Effect of TH-glucan and Conformers on Peripheral Blood Cell Counts Treatment WBC (x 10 6 /ml) RBC (x 10 9 /ml) PLT (x 10 6 /ml) Saline 16.5±0.9 6.44±0.11 512±41 WGP (IP) 17.6±1.1 6.90±0.20** 800±58 WGP (IM) 17.5±1.3 6.64±0.21 922±45 VHMW 17.4±0.7 6.50±0.10 767±18 HTH 25°C 18.7±0.9 7.07±0.18** 670±44 HTH 37 0 C 17.5±0.9 6.30±0.22 672±53 TH-glucan 18.5±0.9 6.51±0.12 494±38 LTH 25°C 19.1±0.7 6.67±0.12 528±36 LTH 37 0 C 20.4±1.2 6.23±0.12 455±47 LMW 18.0±0.8 7.50±0.10** 529±50 percent change vs saline t-test, p <0.05 vs saline.

Effect of TH-glucan and Conformers on Mortality The S. aureus dose used in these studies was a 50% lethal infective dose.

Although all animals were euthanized on day 2 for CFU and blood cell evaluations, a significantly lower mortality was observed in the rats treated with WGP (IP) (p <0.01) and LMW (p 0.05) compared to saline-treated rats. Slightly lower mortality was also seen in the rats treated with WGP (IM) (31% vs saline 48%) and LTH 25 0 C (31% vs saline A slightly higher mortality (63% vs saline 48%) WO 00/13019 PCT/US99/18995 -33was observed in the LTH 37°C-treatment group. However, these differences did not reach statistical significance.

These experiments demonstrated that TH-glucan, as well as conformers (agents which mimic the activity of TH-glucan), can be used to augment host defenses, and thus can be used in the treatment of diseases. It was interesting to note that although the conformer molecular weights changed up to approximately 100 fold VHMW vs LMW), the anti-infective activity remained constant.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any ****:other integer or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

Claims

1. A method of isolating p(1,3)-glucan from a sample, comprising the steps of: a) contacting a sample containing P(1,3)-glucan with a proteinaceous receptor for underivatized aqueous soluble /(1,3)-glucan, under conditions that are suitable for binding of 3(1,3)-glucan to the receptor to form primary complexes of /3(1,3)-glucan bound to receptor; b) isolating the primary complexes; and c) obtaining P(1,3)-glucan from the primary complexes.

2. The method of Claim 1, wherein the proteinaceous receptor is coupled to a solid phase.

3. The method of Claim 2, wherein the solid phase is selected from the group consisting of: filters, membranes, beads, particles, organic resin, microtiter o* plates and slides.

4. A method of isolating P(1,3)-glucan-containing organisms from a sample, comprising the steps of: a) contacting a sample containing P(1,3)-glucan-containing organisms with a proteinaceous receptor for underivatized aqueous soluble /(1,3)-glucan, under conditions that are suitable for binding of 3(1,3)-glucan to the receptor to form 20 primary complexes of /(1,3)-glucan bound to receptor; b) isolating the primary complexes; and c) obtaining P(1,3)-glucan-containing organisms from the primary complexes.

WO 00/13019 PCTIUS99/18995 A method of detecting the presence of P(1,3)-glucan in a sample, comprising the steps of: a) contacting a sample to be tested for the presence of 3(1,3)-glucan with a proteinaceous receptor for underivatized aqueous soluble 3(1,3)-glucan, under conditions that are suitable for binding of 3(1,3)-glucan to the receptor to form primary complexes of 1(1,3)-glucan bound to receptor; and b) detecting the primary complexes formed in step wherein the presence of primary complexes is indicative of the presence of P(l,3)-glucan in the sample.

6. The method of Claim 5, wherein the proteinaceous receptor is attached to a solid phase.

7. The method of Claim 6, wherein the solid phase is selected from the group Sconsisting of: filters, membranes, beads, particles, organic resin, microtiter plates and slides. 5 .9 15

8. The method of Claim 5, wherein the sample is attached to a solid phase.

9. The method of Claim 8, wherein the solid phase is selected from the group consisting of: filters, membranes, beads, particles, organic resin, microtiter plates and slides. o

10. The method of Claim 5, wherein the sample is from an animal source.

11. The method of Claim 10, wherein the sample is a biological fluid.

12. The method of Claim 5, wherein the sample is from a plant source.

13. The method of Claim 5, wherein the sample is from an environmental source.

14. The method of Claim 5, wherein the sample is from a food source.

WO 00/13019 PCT/US99/18995 -36- The method of Claim 5, wherein the sample is from a fermentation source.

16. The method of Claim 5, wherein the primary complexes are detected with a detectable label.

17. The method of Claim 16, wherein the detectable label is selected from the group consisting of: radionucleotides, dyes, fluorescent compounds, biotin and streptavidin.

18. The method of Claim 5, wherein the primary complexes are detected with an antibody.

19. The method of Claim 18, wherein the antibody is an antibody to P(1,3)-glucan. 10

20. The method of Claim 18, wherein the antibody is an antibody to receptor. C

21. A method of detecting the presence of P(1,3)-glucan-containing organisms in a sample, comprising the steps of: Sa) contacting a sample to be tested for the presence of P(1,3)-glucan- containing organisms with a proteinaceous receptor for underivatized aqueous soluble 3(1,3)-glucan, under conditions that are suitable for binding of glucan, to the receptor to form primary complexes of 3(1,3)-glucan-containing organisms bound to receptor; and b) detecting the primary complexes formed in step wherein the presence of primary complexes is indicative of the presence of 20 P(1, 3 )-glucan-containing organisms in the sample.

22. A method of quantifying the amount of P(1,3)-glucan in a sample, comprising the steps of: a) contacting a sample to be tested with a proteinaceous receptor for underivatized aqueous soluble (1,3)-glucan, under conditions that are suitable for binding of /(1,3)-glucan to the receptor to form primary complexes of /(l,3)-glucan bound to receptor; WO 00/13019 PCT/US99/18995 -37- b) isolating the primary complexes formed in step and c) quantifying the primary complexes, wherein the amount of P(1,3)-glucan in the sample correlates with the amount of primary complexes.

23. A method of quantifying the amount of P(1,3)-glucan in a sample, comprising the steps of: a) contacting a sample to be tested with a proteinaceous receptor for underivatized aqueous soluble (1,3)-glucan and a labeled, underivatized, aqueous soluble 0(1,3)-glucan, under conditions that are suitable for binding of 3(1,3)-glucan to the receptor to form primary complexes of underivatized, aqueous soluble /(1,3)-glucan bound to receptor; and b) determining the amount of primary complexes formed in step wherein the amount of P(1,3)-glucan in the sample is inversely related to the amount of primary complexes. 5

24. A method of diagnosing infection by a P(1,3)-glucan-containing organism in an individual, comprising the steps of: a) contacting a sample from the individual with a proteinaceous receptor for underivatized aqueous soluble 3(1,3)-glucan, under conditions that are suitable for 20 binding of 0(1,3)-glucan to the receptor to form primary complexes of 20 glucan bound to receptor; and b) detecting the primary complexes formed in step wherein the presence of primary complexes is indicative of infection by a P(1, 3 )-glucan-containing organism. 4 A kit useful in detecting the presence of P(1,3)-glucan, comprising a proteinaceous receptor for underivatized aqueous soluble (1,3)-glucan and an antibody to underivatized, aqueous soluble, 3(1,3)-glucan.

WO 00/13019 PCT/US99/1 8995 -38-

26. An assay for identifying an agent which has an affinity for a proteinaceous receptor for underivatized, aqueous soluble P(1,3)-glucan, comprising the steps of: a) combining radiolabeled underivatized, aqueous soluble P(1,3)-glucan, receptor for underivatized, aqueous soluble P(1,3)-glucan, and an agent to be tested, under conditions suitable for binding of underivatized, aqueous soluble P(1,3)-glucan to a proteinaceous receptor for underivatized, aqueous soluble P(1,3)-glucan; b) determining the extent of binding of underivatized, aqueous soluble P(1,3)-glucan to the receptor for underivatized, aqueous soluble 3(1,3)- glucan; and c) comparing the extent of binding determined in step with the extent of binding in the absence of the agent to be tested under conditions suitable for binding of underivatized, aqueous soluble P(1,3)-glucan to the receptor for underivatized, aqueous soluble P(1,3)-glucan, wherein a decrease in the extent of binding of underivatized, aqueous soluble P(1,3)-glucan to the receptor for underivatized, aqueous soluble P(1,3)-glucan indicates that the agent has an affinity for the receptor for underivatized, aqueous soluble p(1,3)-glucan.

27. An assay according to Claim 26, wherein the receptor is a compound having an affinity for underivatized, aqueous soluble P(1,3)-glucan.

28. An assay according to Claim 27, wherein the compound is a receptor analogue or a receptor mimic.

29. A method of delivering an agent to a cell containing a proteinaceous receptor for underivatized, aqueous soluble P(1,3)-glucan, comprising the steps of: a) conjugating an agent to be delivered to an underivatized, aqueous soluble P(1,3)-glucan to produce a conjugate molecule; and b) administering the conjugate molecule under conditions suitable for binding of the underivatized, aqueous soluble p(1,3)-glucan of the WO 00/13019 PCTIUS99/18995 -39- conjugate molecule to the receptor for underivatized, aqueous soluble P(1,3)-glucan, whereby the underivatized, aqueous soluble P(1,3)-glucan binds a receptor for underivatized, aqueous soluble P(1,3)-glucan in a cell containing a receptor for underivatized, aqueous soluble P(1,3)-glucan, thereby delivering the agent of the conjugate molecule to the cell.

A method according to Claim 29, wherein the receptor is a compound having an affinity for underivatized, aqueous soluble p(1,3)-glucan.

31. A method according to Claim 30, wherein the compound is a receptor analogue or a receptor mimic.

32. An assay for identifying an agent which alters the effect of underivatized, aqueous soluble P(1,3)-glucan on activation of a signal transduction pathway, said assay comprising the steps of: a) combining underivatized, aqueous soluble P(1,3)-glucan, proteinaceous receptor for underivatized, aqueous soluble P(1,3)-glucan, and an agent to be tested, under conditions suitable for binding of underivatized, aqueous soluble p(1,3)-glucan to the receptor for underivatized, aqueous soluble P(1,3)-glucan; b) determining the extent of activation of a signal transduction pathway; and c) comparing the extent of activation determined in step with the extent of activation in the absence of the agent to be tested under conditions suitable for binding of underivatized, aqueous soluble P(1,3)-glucan to the receptor for underivatized, aqueous soluble P(1,3)-glucan, wherein a difference in the extent of activation of the signal transduction pathway indicates that the agent alters the effect of underivatized, aqueous soluble P(1,3)-glucan on activation of a signal transduction pathway.

33. An assay according to Claim 32, wherein if the extent of activation is greater in the presence of the agent than in the absence of the agent, the agent enhances WO 00/13019 PCT/US99/18995 the effect of underivatized, aqueous soluble P(1,3)-glucan on activation of a signal transduction pathway.

34. An assay according to Claim 32, wherein if the extent of activation is less in the presence of the agent than in the absence of the agent, the agent diminishes the effect of underivatized, aqueous soluble P(1,3)-glucan on activation of a signal transduction pathway.

An assay according to Claim 32, wherein the activation of the signal transduction pathway is measured by the modulation of at least one transcriptional regulatory factor.

36. An assay according to Claim 35, wherein the transcriptional regulatory factor is selected from the group consisting of members of the NF-KB, NF-IL-6 and/or AP-1 families.

37. An assay according to Claim 32, wherein step is carried out by 32 P-labeled DNA oligonucleotides specific for the transcriptional regulatory factor.

3 8. An assay according to Claim 32, wherein the receptor is a compound having an affinity for underivatized, aqueous soluble p(1,3)-glucan.

39. An assay according to Claim3 8 ,wherein the compound is a receptor analogue or a receptor mimic. p.

4 0. A method of activating a signal transduction pathway in a cell containing a proteinaceous receptor for underivatized, aqueous soluble p(1,3)-glucan, comprising: contacting a cell containing a proteinaceous receptor for underivatized, aqueous soluble P(1,3)-glucan with an agent which binds and activates the receptor for underivatized, aqueous soluble P(1,3)-glucan, WO 00/13019 PCT/US99/18995 -41- whereby the receptor for underivatized, aqueous soluble P(1,3)-glucan is activated, thereby activating a signal transduction pathway.

41. A method according to Claim 4 0 pvherein the activation of the signal transduction pathway is measured by the modulation of at least one transcriptional regulatory factor.

4 2. A method according to Claim 41 ,wherein the transcriptional regulatory factor is selected from the group consisting of members of the NF-KB, NF-IL6 and/or AP-1 families.

4 3 A method according to Claim 4 0 ,wherein the agent is underivatized, aqueous soluble 0(1,3)-glucan.

44. A method according to Claim 4 0 Wherein the receptor is a compound having an affinity for underivatized, aqueous soluble p(1,3)-glucan.

45. A method according to Claim 4 4 vherein the compound is a receptor analogue or a receptor mimic.

46. An isolated preparation containing a proteinaceous receptor which binds to a ligand which is an underivatized, aqueous soluble P(1,3)-glucan.

4 7. A preparation according to Claim 4 6,wherein the receptor specifically and s. selectively binds the ligand. S

4 8. A preparation according to Claim 4 6 wherein the underivatized, aqueous soluble P(1,3)-glucan is in a triple helix conformation.

49. Methods, assays or preparations, substantially as hereinbefore described with reference to the Examples. DATED this 18th day of March, 2002 THE COLLABORATIVE GROUP, LTD. By its Patent Attorneys DAVIES COLLISON CAVE