WO2004042052A1 - Modulating tnf (alpha) secretion - Google Patents

Abstract

The invention is directed to characterisation of the steps involved in the intracellular processing, packaging and transport of a cytokine in macrophages and identification of the pivotal points in the pathway that are potentially suitable for genetic and pharmaceutical manipulation. The invention provides a more effective method of reducing or eliminating secretion of a cytokine from a cell. The method further provides a more restricted, tissue-specific elimination of a cytokine, avoiding the problems of inducing immune deficiency, through the identification of cell or tissue-specific molecules to target with drugs. In one form the invention is directed to modulating intracellular trafficking to inhibit or suppress TNFa secretion.

Description

TITLE MODULATING TNF (ALPHA) SECRETION FIELD OF INVENTION THIS INVENTION relates to a method of modulating cytokine secretion in macrophages for the treatment of diseases and disorders resulting from abnormal cytokine secretion. More particularly, this invention relates to a method of inhibition of cytokine secretion through interference with the proteins that facilitate intracellular cytokine trafficking. Particular cytokines include Tumour necrosis factor (TNFα), interleukin-6 and interleukin-10. A particular feature of this invention is the modulation of TNFα secretion through the genetic or pharmaceutical targeting of any regulatory proteins and modulators of the secretory pathway for TNFα. Target proteins include, but are not limited to, syntaxin4₅ muncl8c, Cdc42, Rac-1, VAMP3, syntaxin3, syntaxinό. Vtil-B, Vap3, RGS16 and RGSGAIP. This invention also provides a novel single cell assay which can be used for identifying intracellular trafficking proteins that may be useful targets for the development of drugs that modulate cytokine secretion and for large-scale screening for potential anti-TNF drugs or genes. BACKGROUND OF THE INVENTION Tumour necrosis factor α (TNFα), previously known as cachexin, is one of the main cytokines released from activated macrophages at sites of inflammation. TNFα has many targets and many physiological roles, including cytotoxic, irflammatory. immunological and hematopoietic actions. TNFα is an important pro-inflammatory mediator that primes the immune system by activating and recruiting other cells. At sites of extensive or persistent iriflammation, TNFα is often secreted in excess by large numbers of activated macrophages.

Resting macrophages have low oxygen consumption and little or no cytokine secretion. Upon activation by an appropriate stimulus, macrophages undergo many changes to enact tumour cytotoxic or microbicidal actions.

Amongst the functions initiated during activation is the synthesis and secretion of cytokines including TNFα.

The prototypic activator of macrophages is lipopolysaccharide (LPS) or endotoxin, which is a component of the outer wall of gram-negative bacteria. LPS interacts with serum LPS-binding proteins to form a complex which binds to the glycolipid anchored CD 14 receptor on macrophages (Wright et al, 1990, Science, 249, 1431-1433). CD14 interacts with the recently identified Toll-like Receptor 4 (TLR4), which signals through adaptor proteins, culminating in the expression of downstream inflammatory effector genes (Aderem and Ulevitch, 2000, Nature, 406, 782-787) and the production and secretion of cytokines, such as TNFα and interleukin-1. TNFα is synthesized in macrophages as a 26kd Type II transmembrane precursor which accumulates in the golgi complex, TNFα is then trafficked from the golgi complex to the cell surface where a 17 kd ectodomain is cleaved off by the enzyme TACE. Trimers of this soluble subunit then form the circulating cytokine. TNFα can also be retained on the macrophage surface in an uncleaved form.

Excessive secretion of TNFα has been implicated in several pathologies associated with acute and chronic inflammatory diseases (Beutler, 1999; J. Rheumatol., 26, 16-21; Vassalli, 1992, Annu. Rev. Immunol., 10, 411-452). Excessive or inappropriate secretion of TNFα is one of the leading causes of death in acute conditions such as septic shock, and it is one of the main factors contributing to ongoing tissue damage in chronic inflammatory diseases such as inflammatory bowel disease (IBD), arthritis, psoriasis, congestive heart disease and chronic obstructive pulmonary disease.

In the past, a number of potential anti-TNFα drugs have made it to clinical trials, with varying degrees of success and many notable failures. To date, all of these drugs have targeted extracellular TNFα.

Current anti-TNFα therapies are based on the strategy of sequestering excess TNFα after it has been secreted into tissues and the circulation. The current drugs on the market include a monoclonal antibody to TNFα (Infliximab or Remicade) and a soluble TNFα receptor analogue (Etanercept or Enbrel). Both of these drugs have been approved for use in the treatment of IBD (which encompasses the spectrum of disease characterized as Crohn's disease and ulcerative colitis) with pending approvals for arthritis treatment.

Clinical management of TNFα levels is very important in the management of IBD, and Infliximab has demonstrated positive results in reducing the severity of symptoms and intestinal damage. Infliximab has offered relief particularly to IBD patients who cannot take or are unresponsive to existing steroid-based treatments. Anti-TNFα therapy is therefore increasingly considered necessary and efficacious for the treatment of chronic inflammation.

Current and many previously-tested drugs have been targeted towards the neutralization (or impaired function) of extracellular secreted TNFα and possess the following significant short-comings: (i) they eliminate circulating TNFα throughout the body, thereby comprising immunity, predisposing patients to infection and induce severe side effects, for example, patients being treated with Infliximab have contracted tuberculosis and other infectious and parasitic diseases,

(ii) it is possible that reduced tumour surveillance through the decreased systemic levels of TNFα over the long term might predispose patients to increased frequency of tumours, (iii) the current drugs require repeated adniinistration by injection, and (iv) the drugs are very expensive.

A new alternative strategy for clinical manipulation of TNFα is required. OBJECT OF THE INVENTION The present inventors have realized the current anti-TNFα drugs possess significant short-comings and there is a demonstrated need for the development of new drugs for the treatment of acute and chronic inflammatory diseases. It is therefore an object of the invention to overcome limitations of, or provide an alternative to, the abovementioned background art. A drug or preventative treatment that blocks the secretion of TNFα, so that it never leaves the cell, offers a new approach for anti-TNFα therapies. SUMMARY OF INVENTION

The present inventors have characterised the steps involved in the intracellular processing, packaging and transport of TNFα in macrophages and have identified the pivotal points in the pathway that are potentially suitable for genetic and pharmaceutical manipulation. This invention provides a more effective method of reducing or eliminating secretion of TNFα. Furthermore, by identifying cell or tissue-specific molecules to target with drugs, this invention provides for a more restricted, tissue-specific elimination of TNFα, avoiding the problems of inducing immune deficiency. In one form the invention is therefore broadly directed to the identification of one or more intracellular trafficking proteins involved in the secretion of a cytokine. In another broad form the invention is directed to modulating intracellular trafficking to reduce, suppress, inhibit or block cytokine secretion.

In a first aspect, the invention provides a method of regulating an activity of a pro-inflammatory cytokine including the step of modulating secretion of said cytokine by a cell or tissue of an animal by modulating one or more intracellular trafficking proteins.

The activity of the pro-inflammatory cytokine is regulated in vivo or in vitro. According to this aspect, secretion of the pro-inflammatory cytokine is modulated to thereby modulate an immune response of said animal.

Preferably the cytokine is TNFα, or any other pro-inflammatory cytokine, including, but not limited to, interleukin-6 and interleul in-10.

Preferably the cell is a macrophage. Preferably, the one or more intracellular trafficking protein regulates post- golgi transport of said cytokine.

Preferably, the intracellular trafficking protein(s) are selected from the group consisting of syntaxin4, RGS16, RGSGAIP, RGS, syntaxin3, syntaxin6, Vtil-B, munclδc, Cdc42, VapB and Rac-1. According to this aspect, the invention provides a method of preventing or inhibiting cytokine secretion during the immune response in an animal in response to acute or chronic inflammatory diseases such as septic shock, inflammatory bowel disease, arthritis, psoriasis, congestive heart disease and chronic pulmonary disease.

According to this aspect, the invention also provides a method of priming and/or improving the immune response in an animal in response to infection where increased secretion of TNFα may be advantageous. Preferably, the animal is a mammal. Preferably, the mammal is a human.

In a second aspect, the invention provides a pharmaceutical composition comprising a modulator of pro-inflammatory cytokine secretion which modulates intracellular trafficking of said cytokine.

Preferably, the modulator of pro-inflammatory cytokine secretion is selected from the group consisting of an antibody, peptide, nucleic acid, drug and mimetic.

Preferably, the modulator targets at least one protein selected from the group consisting of syntaxin4, RGS16, RGSGAIP, RGS, syntaxin3, syntaxinό, Vtil-B, munclδc, Cdc42, VapB and Rac-1. More preferably, the pharmaceutical composition comprises an inhibitor of cytokine secretion that targets syntaxin4 and/or muncl8c.

Preferably, the cytokine is TNFα, or any other pro-inflammatory cytokine including, but not limited to, interleukin-6 and interleukin-10.

In a third aspect, the invention provides a method of identifying one or more intracellular trafficking proteins that regulate pro-inflammatory cytokine secretion, said method including the step of identifying one or more proteins that co-localise with said cytokine in a cell, thereby indicating that at least one of said one or more proteins is an intracellular trafficking protein that regulates secretion of said cytokine.

In a fourth aspect, the invention provides a method of identifying one or more intracellular trafficking proteins that regulate pro-inflammatory cytokine secretion, said method including the step of determining whether expression of the one or more intracellular trafficking proteins or nucleic acid encoding same changes in response to stimulation or activation of cytokine production by a cell or tissue, thereby indicating that at least one of said one or more proteins is an intracellular trafficking protein that regulates secretion of said cytokine.

Preferably, expression of the intracellular trafficking protein or nucleic acid encoding same is detected by a microarray technique. Preferably, expression of the intracellular trafficking protein is detected by a protein array technique.

Preferably, said intracellular trafficking protein is detected by immunoblot.

In a fifth aspect, the invention provides a method of identifying a modulator of cytokine secretion, said method including the steps of:

(i) administering a candidate modulator to a cell that secretes a pro- inflammatory cytokine; and (ii) determining whether said candidate modulator affects secretion of said cytokine by said cell. Preferably, the cell is a macrophage.

Preferably, the cytokine is TNFα, or any other pro-inflammatory cytokine including, but not limited to, interleukin-6 and interleukin-10.

In a sixth aspect, the invention provides a use of an intracellular trafficking protein for identification, production, or design of a modulator of pro- iriflammatory cytokine secretion.

Preferably, the intracellular trafficking protein is selected from the group consisting of syntaxin4, RGS 16, RGSGAIP, RGS, syntaxin3, syntaxinό, Vtil-B, munclδc, Cdc42, VapB and Rac-1. Preferably, the pro-inflammatory cytokine is selected from the group consisting of TNFα, interleukin-6 and interleukin-10.

Throughout this specification, "comprise", "comprises" and "comprising" are used inclusively rather than exclusively, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES FIG. 1 : The change in SNARE protein levels in response to LPS-activation.

A. Cytosol (C) and total microsomal membranes (M) (2 μg protein/lane), prepared from cultured RAW264 cells after treatment for 0, 2 or 24 hours with LPS (100 ng/ml), were analyzed by SDS-PAGE and immunoblotting using a panel of antibodies to detect syntaxins (Syn) and the other proteins indicated. Protein staining of actin is included as a loading control.

B. Average densitometric values from immunoblots (n=4) were used to calculate the relative amount of each protein at 0, 2 and 24 hours after stimulation with LPS (100 ng/ml). The fold changes in some of the cell surface-associated SNARE and secl-munc like (SM) proteins are shown in the first graph. In the second graph, the expression patterns of syntaxins in exocytic (solid symbols) and endocytic (open symbols) pathways are compared.

C. Immunofluorescence staining of endogenous syntaxin4 on fixed, permeablized RAW264 cells showing brighter staining of syntaxin4 on the surface of cells after 2 hours of LPS (100 ng/ml) treatment as compared to the 0 and 24 hour time points. D. Cell homogenates were fractionated to produce fractions enriched for high density membranes (HDM), low density membranes (LDM), plasma membrane (PM) and cytosol (CYT) and which were then separated on

SDS-PAGE. Immunoblotting with specific antibodies detected munclδc and syntaxin4 together in plasma membrane and high density membranes while a control protein, GAIP, a vesicle-associated protein, was enriched in low density membranes.

. E. Muncl 8c was immunoprecipitated from cell extracts and the proteins in the supernatant (SN) and immunoprecipitate (IP) were analyzed by immunoblotting. Syntaxin4 but not syntaxin2 coimmunoprecipitates with muncl 8c in macrophages.

FIG. 2: Cell surface delivery of TNFα in activated macrophages.

A. Immunofluorescence staining of TNFα in fixed, permeabilized RAW264 cells shows the accumulation of newly-synthesized TNFα in the perinuclear golgi complex 2 hours after activation with LPS (100 ng/ml). By 24 hours, TNFα synthesis has ceased.

B. Staining of fixed unpermeabilized cells after treatment with LPS (100 ng/ml) for 1 hour in the absence or presence of a TACE inhibitor (upper 2 panels). Newly synthesized TNFα is normally cleaved rapidly from the cell surface, where it is then difficult to detect; the inhibitor blocks this cleavage and results in retention and increased surface staining of TNFα. The single cell assay for cell surface delivery of TNFα in cells treated with LPS and inhibitor is shown in the lower 3 panels; cells are stained sequentially to detect surface TNFα on unpermeabilized cells (left hand panel), followed by permeabilization and staining for an intracellular protein, in this case the golgi- vesicle-associated protein γ-adaptin (right hand panel). FIG. 3: The t-SNARE syntaxin4 regulates cell surface delivery of TNFα. A. Cells were microinjected with fusion proteins corresponding to the cytoplasmic tails of syntaxin2 and syntaxin4 plus GST alone (GST- syn2tail, GST-syn4tail, GST alone), diluted in microiηjection buffer (10 mMKH2PO4, pH 7.2, containing 10 mM KC1 and Texas Red conjugated dextran to mark the injected cells) to a final concentration of approximately lmg/ml. After 5 hours of recovery cells were treated with

LPS (100 ng/ml) and TACE inhibitor, then immunostained for surface TNFα. Arrow heads denote the injected cells. The levels of surface TNFα staining in microinjected cells, depicted by the Texas Red staining, were compared to surrounding iininjected cells. Images were analysed to measure pixel intensity within a defined area at the cell surface and the percentage of cells where the surface staining level is reduced by 3 fold or more compared to uninjected cells is pepicted in the bar graph. Results in the graph represent standard errors of the mean. Each data point calculated from data collected from 50 injected cells per condition in 3 experiments.

B. Muncl 8c-GFP cDNA (10 μg) was introduced into RAW264 macrophage cells by electroporation and the next day cells were treated with TACE inhibitor and LPS (100 ng/ml), to induce TNFα synthesis, for one hour prior to fixation. Unpermeabilized cells were immunostained to detect surface TNFα which was then assessed on GFP-labeled cells (denoted with an arrow head) compared to surrounding cells. These images are representative of > 50 fields containing transfected cells in each of three separate experiments. C. cDNA (10 μg) encoding HA-tagged syntaxin 4 was electroporated into

RAW264 macrophage cells which were then treated with LPS (100 ng/ml) and TACE inhibitor. Unpermeabilized cells were immunostained to detect surface TNFα then cells were permeabilized and stained with an HA antibody. TNFα surface staining was compared in cells expressing HA-syntaxin4 and in untransfected cells in three replicate experiments.

Images were analysed to measure pixel intensity within a defined area at the cell surface and the percentage of cells where the surface staining level is increased by 2 fold or more compared to uninjected cells is depicted in the bar graph. Results in the graph represent standard errors of the mean. Each data point calculated from data collected from 50 injected cells per condition in 3 experiments. FIG. 4: Microarray analysis of trafficking protein gene expression in response to LPS. Gene transcripts of several families of proteins known to be involved in regulating trafficking that change after 2 hours of treatment with LPS (100 ng/ml) in RAW264 macrophages as compared to untreated cells were selected from the array data for display on this graph. Overall gene expression changes show significant up (black lines above the x- axis) or down (grey lines below the x-axis) regulation of multiple genes at 2 hours (coincident with the peak expression of TNFα mRNA itself - dotted line) and at 12 hours post-LPS. FIG. 5: Microarray analysis of trafficking protein gene expression in response to LPS. Gene transcripts of several families of proteins known to be involved in regulating trafficking that change after 2 hours of treatment with LPS (100 ng/ml) in RAW264 macrophages as compared to untreated cells were selected from the array data for display on this graph. Changes in gene expression levels in RAW264 macrophages after addition of LPS (100 ng/ml) of specific trafficking proteins are shown. All responses recorded here are assessed as significant changes in the context of the Affymetrix system. Candidates selected from this analysis will be studied further as potential drug targets for regulating TNFα frafficking. FIG. 6: Identification of proteins regulated by LPS. RAW264 cells were stimulated for 0, 0.5, 2, 7, 12 and 24 hours with LPS (100 ng/ml), then lysed. Cell extract samples (100 μg total protein /lane) were analysed by SDS-PAGE and immunoblotting to detect TNFα, syntaxins 3 and 6, Vtil- B, SNAP-23, Munc-18c and GAIP. These trafficking proteins showed upregulation of protein levels between 2 and 7 hours. TABLE 1 : Delivery of newly-synthesized TNFα to the cell surface in macrophages and effects of specific drugs. In each case, drugs were added to macrophages simultaneously with LPS and therefore act on post- golgi trafficking.

DETAILED DESCRIPTION OF INVENTION The present inventors have characterised steps in the pathway involved in the intracellular transport and secretion of TNFα in macrophages and identified individual trafficking proteins and protein complexes as potential targets for pharmaceutical and genetic manipulation.

The trafficking proteins are involved with, are associated with, control or otherwise regulate cytokine secretion from a cell. Previous studies have concentrated on the areas of immunology and endocrinology where the main focus has been the processing and function of extracellular TNFα. Therefore drug development has targeted post-secretion extracellular TNFα or the enzymes that cleave TNFα off the macrophage cell surface. In contrast, the present invention has resulted in the characterization of the intracellular regulation of TNFα secretion and their studies have revealed novel targets for drug intervention.

By "intracellular trafficking^^'' is meant the processes by which a cytokine is secreted including one or more of:

(i) loading of the vesicles with a cytokine (ii) intracellular transport of vesicles from the endoplasmic reticulum to the plasma membrane via intervening compartments (iii) docking of the vesicles at the plasma membrane (iv) fusion of the vesicles with the plasma membrane, and (v) exocytosis

N-ethylmaleimide factor (NSF), syntaxin, VAMP and SNAP proteins are but a few of the proteins which form high molecular weight complexes to drive vesicle docking and fusion with the plasma membrane or with other membranes. A vesicle-associated protein complex (v-SNARE) binds to SNAP receptor (t- SNARE) complexes located on the plasma membrane and the 4 helix bundle formed as a result of trans-SNARE pairing is responsible for the specificity of docking and the subsequent completion of membrane fusion. Syntaxins generally form part of the t-SNARE complex; different syntaxins are distributed on different membrane domains to provide for the specificity of SNARE-mediated docking and fusion. SNARE proteins are essential components of the exocytotic machinery because it has been demonstrated that cleavage of specific SNARE proteins (for example by botulinum toxin Cl) results in an inhibition of protein secretion. The inventors found that syntaxins and other SNARE-related proteins, in particular, are regulated by LPS in macrophages. Screens for changes in protein levels and gene expression levels, developed by the inventors to monitor the LPS-activated trafficking proteins in macrophages, surprisingly revealed that many of these proteins are regulated upon activation of the macrophages with LPS. At the protein level the inventors discovered that cellular levels of the syntaxins typically involved in exocytotic pathways (syntaxins 2, 4 and 6) were increased by LPS-activation, whereas the syntaxins generally involved in endocytosis (syntaxins 7, 11, and 13) were decreased or unaffected by LPS- activation.

A finding of particular interest was that the increased expression of syntaxin4 temporally matched the increased expression of TNFα. The levels of other t-SNARE proteins; SNAP23, muncl 8c (SM) protein and muncl 8c were also significantly increased after LPS stimulation. Syntaxin4 and muncl 8c were demonstrated to cofractionate and to be mostly associated with the plasma membrane. It was also observed that muncl 8c and syntaxin4, and SNAP23 and syntaxin4 form, stable complexes in macrophages. When muncl 8c was immunoprecipitated from LPS-activated macrophages, syntaxin4 but not syntaxin2 was coprecipitated. Therefore, syntaxin4, muncl 8c and SNAP23 form a specific t-SNARE complex in macrophages at the plasma level.

The inventors also have developed a single cell immunofluorescence- based assay, which monitors the intracellular trafficking of TNFα and can therefore be used to determine the function of specific intracellular regulators or molecules. The intracellular regulators or molecules can be targeted or manipulated by specific drugs, and the microinjection of peptides, antibodies, cDNAs, mRNAs or double stranded RNA in the assay. The assay is a useful tool for identifying molecules with the capacity to block, prevent, suppress or otherwise inhibit TNFα secretion. Throughout this specification reference is made to TNFα, IL-6 and IL-10 but it will be appreciated that the broad principles described here may also to apply to other cytokines in terms of the broad applicability of concept and principle. By "cytokine" is meant a mediator released by cells of the immune system that acts as an intercellular mediator in the generation of an immune response. Preferred cytokines are pro-inflammatory cytokines including but not limited to TNFα, interleukin-1, interleukin-6 and interleukin-10.

By "pro-inflammatory cytokine" is meant a cytokine that plays a role or has some involvement in an inflammatory process or inflammatory response.

For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

By "protein" is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids D- and L- amino acids, as are well understood in the art.

A "peptide" is a protein having no more than fifty (50) amino acids. A "polypeptide" is a protein having more than fifty (50) amino acids. The term "nucleic acid"¹ as used herein designates single-or double- stranded mRNA, RNA, cRNA, RNAi and DNA inclusive of cDNA and genomic DNA.

Identification of intracellular trafficking proteins The invention contemplates identification of intracellular trafficking proteins, in particular, proteins involved with intracellular trafficking of a pro- inflammatory cytokine. Identification of said proteins may be achieved by detecting nucleic acids encoding the intracellular trafficking proteins, or detecting the proteins per se. A change in nucleic acid expression, for example, an increase or decrease, may correlate with increased or decreased production or translation of a protein. Hereinafter are provided examples of methods for detecting nucleic acid or protein expression. Preferably, a change in respective nucleic acid or protein expression levels in response to stimulation or activation of cytokine production may identify a protein that regulates said pro-inflammatory cytokine.

The method in one form includes the step of identifying one or more intracellular proteins that co-localise with a cytokine in a cell, for example, one or more intracellular frafficking proteins co-localised with TNFα. This gives an indication of a role for the intracellular protein(s) in regulating cytokine activity. It will be appreciated that these methods may be used to investigate the role of or detect a new intracellular protein. Nucleic acid-based detection

In one form, the invention utilizes a molecular library in the form of a nucleic acid array that comprises a substrate to which is immobilized, bound or otherwise coupled a plurality of nucleic acids encoding trafficking proteins described herein, or respective fragments thereof. Each immobilized, bound or otherwise coupled nucleic acid has an "address" on the array that signifies the location and identity of said nucleic acid. Nucleic acid array technology has become well known in the art and examples of methods applicable to array technology are provided in Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons NY USA 1995-2001).

The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm, and ranges therebetween. The substrate may be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to the trafficking protein-associated nucleic acids of the invention may also be disposed on the array. In certain embodiments, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a member of a nucleic acid library, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for a nucleic acid library member. Each address of the subset can include a capture probe that hybridizes to a different region of a library member.

With respect to the present invention, a preferred nucleic array format is described in more detail hereinafter.

An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

It will be appreciated that nucleic acids may be attached directly or indirectly to other suitable substrates, such as beads, that are appropriate for nucleic acid identification as is well known in the art.

Nucleic acid sequence amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 15 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in International Application WO 92/01813 and International Application WO 97/19193; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et α/.,1994, Biotechniques 17 1077; and Q-β replicase amplification as for example described by Tyagi et al, 1996, Proc. Natl. Acad. Sci. USA 93 5395.

A preferred nucleic acid sequence amplification technique is PCR.

As used herein, an "amplification product refers to a nucleic acid product generated by nucleic acid amplification techniques.

In one particular form the invention contemplates quantitative PCR using primers corresponding to quantify relative expression levels of nucleic acids that encode the intracellular trafficking proteins.

PCR amplification is not linear and hence end point analysis does not always allow for the accurate determination of nucleic acid expression levels.

Real-time PCR analysis provides a high throughput means of measuring gene expression levels. It uses specific primers, an intercalating fluorescent dye such as S YBR Green I or ethidium bromide (EtBr) and fluorescence detection to measure the amount of product after each cycle. Hydridization probes utilise either quencher dyes or fluorescence directly to generate a signal.

An even more preferred method includes real-time PCR analysis. The expression levels of the nucleic acids encoding the trafficking proteins can alternatively be quantified using hybridization and blotting techniques.

"Hybridise and Hybridisation" is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA, RNA-

RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing between complementary purines and pyrimidines as are well known in the art.

Northern blotting is used to identify a complementary RNA sequence. Typically, complementary nucleotide sequences are identified by blotting techniques that include a step whereby nucleotides are immobilised on a matrix (preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step. Dot blotting and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNA polynucleotide sequences. Such techniques are well known by those skilled in the art, and have been described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Ausubel et al, pages 2.9.1 through 2.9.20. Protein-based detection

In one embodiment, trafficking proteins may be detected in a protein library displayed in a number of ways, e.g., in phage display or cell display systems, in protein arrays or by two-dimensional gel electrophoresis, or more specifically, differential two-dimensional gel electrophoresis (2D-DIGE). These particular embodiments may generally be referred to as "proteomic" or "protein profiling" methods, such as described in Chapters 3.9.1 and 22 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, John Wiley & Sons NY USA (1996-2002). Preferably, the protein array comprises a substrate to which is immobilized, impregnated, bound or otherwise coupled a plurality proteins or respective fragments thereof. Each immobilized, impregnated bound or otherwise coupled protein is at an "address" on the array that signifies the location and identity of each said protein or fragment. The substrate may be a chemically-derivatized alrjminium chip, a synthetic membrane such as PVDF or nitrocellulose, a glass slide or microtiter plates.

In another embodiment detection of intracellular trafficking proteins or peptides, may be performed using immunodiagnostic detection. Immunodiagnostic detection may be performed by any of a number of techniques, such as immunoblotting, immunochromatography, Enzyme-Linked Immunosorbent Assay (ELISA), fluorescence microscopy and immimohistochemistry, as are well known in the art. A detailed discussion of ELISA can be found in Unit 11.2, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-1999). A preferred ELISA method is described in the Examples hereinafter. A detailed discussion of Western blot, or immunoblot, can be found in

Unit 10.8, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-1999), and will be described in more detail hereinafter.

In a particularly preferred embodiment, the invention provides a method of identifying one or more intracellular proteins involved in the trafficking of a pro-inflammatory cytokine, such as TNFα, interleukin-6 and interleukin-10. In a particular preferred form, the method involves activating macrophages with LPS and a TNFα converting enzyme (TACE) inhibitor, fixing the cells for 1 hour, staining TNFα on the cell surface with an antibody, permeabilising cells with a detergent and restaining the cells with antibodies directed to intracellular TNFα and intracellular frafficking proteins of interest. Cells are observed under fluorescence microscopy to monitor the levels of staining, in particular the cell surface staining of TNFα before and after manipulation of specific intracellular proteins. Modulators of pro-inflammatory cytokine secretion

The present invention provides methods of modulating secretion of pro- inflammatory cytokines. A modulator of cytokine secretion can be a molecule or substance that affects the amount of cytokine secreted by macrophages. Modulators of cytokine secretion could inhibit, augment, interact or interfere with one or more proteins or steps involved with cytokine production or secretion. Said modulator may interact or interfere with the synthesis of cytokine or a protein involved in the secretory process, or disrupt exocytosis or protein trafficking from the endoplasmic reticulum to the plasma membrane.

The term "target" herein comprises: interact, bind, cleave, block, antagonize, inhibit, agonise, activate or regulate.

TNFα and IL-6 are both early response pro-inflammatory cytokines that are secreted at the same time. It is therefore likely that modulators identified experimentally for manipulating TNFα secretion will also modulate IL-6 secretion. IL-10 is a late response cytokine and since some of the trafficking proteins identified herein are also temporally regulated at later times coincident with IL-10, it is likely they could be used as modulators for this cvtokine.

The modulators of cytokine secretion could be agonists, antagonists, mimetics or modulatory nucleic acids. Such molecules may be proteins, peptides, small organic molecules, or nucleic acids such as antisense oligonucleotides or RNAi.

For example, modulators of cytokine secretion may be identified by way of screening libraries of molecules such as synthetic chemical libraries, including combinatorial libraries, by methods such as described in Nestler & Liu, 1998,

Comb. Chem. High Throughput Screen. 1, 113 and Kirkpatrick et al, 1999,

Comb. Chem. High Throughput Screen 2, 211. It is also contemplated that libraries of naturally-occurring molecules may be screened by methodology such as reviewed in Kolb, 1998, Prog. Drug. Res. 51, 185.

More rational approaches to designing modulators as described herein may employ computer assisted screening of structural databases, computer- assisted modelling, or more traditional biophysical techniques which detect molecular binding interactions, as are well known in the art.

Computer-assisted structural database searching is becoming increasingly utilized as a procedure for engineering agonists and antagonist molecules. Examples of database searching methods may be found in International

Publication WO 94/18232 (directed to producing HIV antigen mimetics), United

States Patent No. 5,752,019 and International Publication WO 97/41526 (directed to identifying EPO mimetics), each of which is incorporated herein by reference.

Generally, other applicable methods include any of a variety of biophysical techniques which identify molecular interactions. Methods applicable to potentially useful techniques such as competitive radioligand binding assays, analytical ultracentrifugation, microcalorimetry, surface plasmon resonance and optical biosensor-based methods are provided in Chapter 20 of CURRENT

PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, 1997) which is incorporated herein by reference.

In a particular embodiment, the present inventors contemplate structural determination of the specific proteins involved in TNFα secretion, for example syntaxin4 and muncl 8c. Nucleic acid modulators

The invention also relates to nucleic acid modulation of intracellular trafficking proteins. Nucleic acid modulators may be any nucleic acid that suppresses, inhibits or reduces pro-inflammatory cytokine secretion by targeting expression of intracellular frafficking proteins.

For example, RNA interference (RNAi) is a novel method of post- transcriptional gene silencing (Hannon, 2002, Nature, 418, 244-251) and involves the introduction of double-stranded RNA into a cell to silence a specific gene. The double-stranded RNA triggers depletion of the targeted rnRNA sequences in the cell without effects on the rate of transcription. This technology may provide a method to prevent, inhibit, attenuate or suppress the production of intracellular proteins involved in cytokine secretion in macrophages, such as syntaxin4 and muncl 8.

The targets for drug and genetic manipulation of the present invention are intracellular targets. Therefore it may be necessary to devise and utilise methods of delivering a modulator into the macrophage cytoplasm. Examples of methods used to deliver a modulator into the macrophage cytoplasm include but are not limited to:

(i) engineering a peptide or antibody that can be delivered into a macrophage via a specific receptor;

(ii) the use of peptide, small molecule or antibody coated beads that can be ingested by the macrophage; and (iii) the use of toxins, such as tetanus toxin to manipulate the functions of syntaxins or VAMPs and botulinum toxin to manipulate Rho GTPases, such as Rac-1 and Cd42.

Examples of techniques that might be applicable to the delivery of oligonucleotides and antisense oligonucleotides into targeted cells are provided in

Guillem et al, 2002, J. Control Release, 83, 133, and Hu et al, Nucleic Acids Res., 2002, 30, 3632, 41. Pharmaceutical compositions

The invention also provides pharmaceutical compositions that comprise at least one modulator of cytokine secretion, for example, as described above.

With regard to TNFα, pharmaceutical compositions are suitable for the therapeutic and prophylactic treatment of diseases or disorders resulting from the abnormal secretion of TNFα, such as septic shock, inflammatory bowel disease, arthritis, psoriasis, congestive heart disease and chronic pulmonary disease. Suitably, the pharmaceutical composition comprises an appropriate pharmaceutically-acceptable carrier, diluent or excipient.

By "pharmaceutically-acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.

A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference.

Any safe route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Infra-muscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present invention, should be sufficient to affect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

So that the present invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting examples.

EXAMPLES Materials and Methods

Cell culture

RAW264 murine macrophages were maintained at 37°C in a 5% CO₂ incubator and grown in RPMI 1640 medium (Biowhittaker, Walkersville, Maryland) supplemented with 10% heat inactivated (56°C, 30 mins) serum supreme (BioWhittaker) and 1% L-glutamine (GIBCO, Grand Island, New

York). Cells were harvested every second day from 85 mm bacterial dishes using a 181/2 gauge syringe and passaged at low concentration in a new dish. Drug and temperature treatments

Macrophages were activated by incubating cells grown on coverslips or on tissue culture dishes with lipopolysaccharide (LPS; 100 ng/ml) (from

Salmonella Minnesota Re595) and in some experiments with interferon γ (IFNγ;

500 pg/ml) (provided by Kate Stacey, 1MB), added to their medium at 37°C for various lengths of time. Other drug treatments used during this project involved incubating cells in medium containing the following: BB3103 metalloprotease inhibitor (10 μM) (from British Biotech Pharmacueticals, Oxford, United Kingdom); Cycloheximide (CHX; 10 μg/ml); (3-methyl-cyclodextrin (cyclodextrin; 10 μM); Jasplakinolide (Jas; 2 μM) (Molecular Probes, Oregon, USA), Cytochalasin D (Cyt D; 10 μM). Unless stated, these drugs were purchased from Sigma Chemical Company, Sydney, Australia. 20°C temperature block: Cells were incubated at 20°C in media containing 10 mM HEPES for 2 hrs to block the trafficking of proteins through the secretory pathway, followed by 30 min incubation at 37°C to allow post golgi trafficking to resume. Immunoflourescence staining

RAW264 cells were plated at low density and grown for 2 days on coverslips. Cells were fixed in 4% paraformadehyde for 60 mins and permeabilised for 4 mins in 0.1% Triton X-100 in PBS. Coverslips were incubated with 30 μl Rl drops of diluted primary antibodies for 1 hour followed by Alexa, or Cy3 -conjugated secondary antibodies (Jackson Labs and Molecular probes, respectively) using blocking washes of 0.5% BSA in PBS. For some experiments, cells were additionally stained with Texas-red or Alexa-conjugated phalloidin or with DAPI (Molecular Probes). Coverslips were mounted on glass slides in 50% glycerol and 1% n-propyl gallate in PBS and sealed with nail enamel. Staining was examined using an Olympus Provis epifluorescence AX70 microscope and digital images were collected on a Dage 300E100T-RC 0.5" cooled CCD camera with a SCION LG-3 frame-grabbing card using NIH software. Single cell assay to detect secretion of TNFα by immunofluorescence

An assay to detect the cell surface delivery and secretion of TNFα in single cells was developed. The assay is based on immunostaining of TNFα in the golgi and then at the cell surface during a defined period (1 hour) after initiating its synthesis and trafficking by treating macrophages with LPS. TNFα is normally released very rapidly from the cell surface by proteolytic cleavage. To retain surface TNFα and optimize its detection the protease inhibitor, BB3103, was added to cells along with LPS to inhibit the processing and release of TNFα. After fixation, TNFα on the cell surface was maximally detected by staining unpermeabilised cells (Fig.2). Visualisation of intracellular TNFα in the golgi complex or to detect intracellular protein of interest or reagents is carried out by permeablising cells and restaining with appropriate antibodies or markers. This assay provided the means to assess TNFα delivery to the cell surface in individual cells transfected or microinjected with specific cDNAs or reagents or in whole populations of cells treated with a drug.

SDS-PAGE and Immunoblotting

Proteins were separated by SDS-PAGE on Laemmli denaturing gels under reducing conditions. Samples were prepared in Laemmli sample buffer (30 mM Tris, 1% SDS, 12% glycerol, 15% P-mercaptoethanol, pH 6.8) and boiled for 5 mins before loading on 12% mini-gels (Biorad) oron 5-15% gradient polyacrylamide standard gels. Mini-gels were run for 50 minutes at 200 V while standard size gels were run for 5 hrs at 40 mA or overnight at 10 mA in running buffer containing 25 mM Tris, 192 mM glycine and 0.1 % SDS. Proteins on the gels were transferred onto PVDF Immobilon P membrane (Millipore, Bedford, Massachusetts) in transfer buffer (0.015 M Tris, 0.12 M glycine, 20% Methanol). Mini-gels were transferred at 120 V for 60 minutes, while standard gels were transferred for 5 hours at 300 mA or overnight at 150 mA. To check protein loading, membranes were stained in 0.1 % Commassie

Brilliant Blue 8250 (in 50% methanol and 10% acetic acid) and destained in 50% Methanol, 10% acetic acid.

For immunoblotting, membranes were reactivated in methanol, washed in Blotto (20 mM Tris-HCl buffer pH 7.4 with 0.15 M NaCl, 0.1% Triton X-100 and 5% nonfat milk powder) and then incubated with agitation for 2 hrs at room temperature or overnight at 4°C in primary antibody diluted in Blotto, followed by further Blotto washes. Membranes were then incubated for 1 hour with a horse-radish peroxidase-conjugated secondary antibody (AMRAD). Detection of immunolabelled protein was with ECL Western Blotting detection reagent (Amersham International, Buckinghamshire, United Kingdom). NIH image analysis software was used to quantitate the intensity of bands. Subcellular fractionation

Cells grown in 30 cm tissue culture dishes were washed three times in homogenization buffer (10 mM Tris, 1 nM EDTA, pH 7.4) with protease inhibitors (Complete protease inhibitor cocktail tablets, Boehringer Mannheim).

Cells were scraped into 2 ml of homogenisation buffer using a rubber policeman, incubated for 5 rnins on ice, then drawn through a 27.5 gauge needle 20 times to lyse cells. Cells were centrifuged at 4°C for 10 minutes at 2 000 rpm and the postnuclear supernatant was then fractionated into total microsomal membranes (pellet) and cytosol (supernatant) by ultracentrifugation at 45 000 rpm, 4°C for 90 minutes. The cytosol fraction was recentrifuged at 45 000 rpm before being aliquoted. Membrane pellets were resuspended in 200 μl of homogenisation buffer containing protease inhibitors. Samples were either snap frozen and stored at -70°C or immediately solubilised in sample buffer and boiled. Protein concentrations were determined using the Pierce BCA protein assay kit. Immunoprecipitation of munclδc

Protein A-Sepharose beads (Pierce, Rockford, Illinois) were rinsed with PBS and incubated with 3 μg of Munclδc antiserum at 4 °C overnight on a rotatory shaker. Fresh membranes were prepared from macrophages as described above. The membrane pellet was resuspended in 100 μl of HES buffer (20 mM Hepes, 1 mM EDTA, 250 mM sucrose) with 1% Triton X-100 and protease inhibitors and left on ice for 25 minutes, then centrifuged at 4°C at 14 000 rpm. The supernatant was pre-cleared for 1 hr at 4°C with protein A-sepharose to eliminate non-specific IgG and proteins binding non-specifically to the beads. The membrane extract was then incubated with coated beads for 2 hrs at 4°C on a rotatory shaker. The beads were collected by brief centrifugation, washed and solubilised in 20 μl of SDS-PAGE sample buffer containing DTT (Boehringer, Mannheim). The supernatants were resolved by 12% SDS-PAGE gel and blotted using polyclonal antibodies to munclδc, syntaxin4 and syntaxin2. Sucrose flotation gradient

For flotation analysis, 600 μg of total cell membrane was solubilised at 4°C for 10 mins in lysis buffer (10 mM Tris-HCI, 150 mM NaCl, 5 mM EDTA, pH 7.5) containing 1% Triton X-100. The sample was then brought to a final sucrose concentration of 45% and final volume of 600 μl in a Beckman 11 x 34 mm tube. This sample was then sequentially overlayed with 7 x 228 μl layers of sucrose to form a decreasing sucrose gradient (35%, 30%, 25%, 20%, 15%, 10%, 5%) and spun for 18 hrs at 50 000 rpm (4°C) in a Beckman TLS55 rotor. Ten 200 μl fractions were collected starting from the top of the gradient, solubilised in sample buffer then separated on a 5%-15% gradient gel and western blotted for syntaxin4, SNAP23, munclδc and Flotillin. Electroporation

Cells in exponential growth were harvested by pelleting, washed and resuspended at 2.5 x 10⁷ cells/ml in serum free media. An aliquot (250 μl) of this cell suspension was electroporated at room temperature with 10 μg DNA. Electroporation was performed with a Gene Pulser ® II (BIORAD) at high capacitance setting (280 mV and 950 μF). Cells were split at a ratio of 1 :2 into fresh growth medium. Immunofluorescence staining was performed 24 hrs later. A variety of wild-type or mutant cDNA plasmids were used in experiments. Most of these were expressed in pcDNA3.1 mammalian expression vectors driven by a CMV based promoter. Microarray analysis

Mouse macrophages were incubated in the absence or presence of LPS for various times between 30 mins and 24 hours. RNA was extracted from cells using the RNAqueous RNA purification kit (Ambion) at each time point and used to produce double stranded cDNA. The cDNA was subsequently used to make biotin labeled cRNA according to the standard Affymetrix protocol. The fragmented biotinylated probe was hybridised to the Affymetrix mouse MOE 430 A chip, which has more than 39,000 transcripts and variants, including over 34,000 well-substantiated mouse genes. Typically each of the genes is represented by a set of 11-20 probe pairs, each consisting of a perfect match and a one-base, centrally-positioned mismatch. The Affymetrix microarray analysis suite software was used to determine gene transcript levels. To compare genes from chip to chip global scaling was used. Three measures of gene expression were used, absolute call, average difference and change call. For the absolute call each gene is assigned a call of present, absent or marginal, while the average difference is calculated by taking the difference between the perfect match and mismatch probe pairs and averaging the difference over the entire probe set. The change call assesses probe pair saturation, calculates a change p-value, and assigns an increase, decrease, no change and a marginal increase or decrease call.

Example 1 Figure 1 shows the change in SNARE protein levels in response to LPS- activation. The LPS-activated increased expression of syntaxin4 temporally matched that of TNFα in which there is a > 2.5 fold increase in cell-associated protein levels at 2 hours post LPS-activation. Syntaxin4 is known to operate in conjunction with another t-SNARE, SNAP23, and with the Seclp-like/muncl8c (SM) protein, muncl 8c. The levels of these proteins were also significantly increased 2 hours after LPS stimulation (Fig. 1).

Cellular levels of syntaxins typically involved in exocytotic pathways (syntaxins 2, 4 and 6) were increased by LPS-activation, whereas the syntaxins generally involved in endocytosis (syntaxins 7, 11, and 13) were decreased or unaffected by LPS-activation (Figs. 1 A, B).

Changes were observed in the levels of other SNAREs or SM proteins, but often with different temporal responses. For example, muncl 8b, an SM protein previously shown to regulate vesicle transport to the apical membrane in polarized cells, was elevated at 24 h after activation (Fig. 1).

The increased expression of individual syntaxins and SM proteins was also seen by immunofluorescence staining of fixed cells at the various time points. Immunostaining of endogenous syntaxin4 (Fig 1C) and of overexpressed GFP-muncl8c (Fig 3B) showed both proteins are primarily located at the cell surface of macrophages, as they are in other cell types. Increased staining intensity for syntaxin4 on the cell surface of macrophages accompanied its increased expression levels at 2 hours post LPS-activation (Fig. 1C). There was generally no change in the targeting of syntaxins or SM proteins to the cell membrane in response to LPS, despite their increased protein levels. A crude fractionation scheme for separating a plasma membrane-enriched fraction from low and high density membranes, also showed that endogenous syntaxin4 and muncl 8c cofractionate and are mostly associated with plasma membrane (Fig. ID). The parallel changes in the levels of syntaxin4, SNAP23 and muncl 8c in macrophages in response to LPS support a closely-linked or common function for these proteins. Consistent with this idea we observed that muncl 8c and syntaxin4 (Fig. IE), and SNAP23 and syntaxin4 (data not shown) form stable complexes in macrophages. When muncl 8c was immunoprecipitated from LPS-activated macrophages, syntaxin4 but not syntaxin2 was coprecipitated (Fig. IE). Therefore, syntaxin4, muncl 8c and SNAP23 form a specific t-SNARE complex in macrophages.

Example 2 Experiments were conducted to determine if syntaxin4 was the t-SNARE that controls TNFα secretion in macrophages. A single cell functional assay to

monitor TNFα delivery to the cell surface in individual microinjected or transfected macrophages was developed.

Newly-synthesized pro-TNFα (a Type II membrane protein) accumulates in the golgi complex after LPS-activation where staining persists at high levels for 4-6 hrs and thereafter diminishes with cessation of TNFα synthesis (Fig. 2A).

Normally, when TNFα reaches the cell surface it is cleaved rapidly by the

protease TACE (TNFα converting enzyme) to release a 17 kDa soluble ectodomain, leaving little detectable surface staining of TNFα (Fig. 2). Addition of a TACE inhibitor blocks the cleavage of TNFα and retains newly-synthesized TNFα at the cell surface where it is maximally immunostained on fixed, unpermeabilized cells (Fig. 2B). Cells can then be permeabihzed to immunostain intracellular, endogenous or co-expressed proteins (Fig. 2B, 3B and 3C).

The assay was conducted to measure exocytosis of TNFα after blocking the function of syntaxin4. Syntaxin mutants in which the fransmembrane domain has been deleted have dominant inhibitory effects on membrane trafficking. Macrophages were microinjected with recombinant fusion proteins corresponding to GST alone or GST fused to the cytoplasmic tails of either syntaxin2 or syntaxin4. Cells were then stimulated with LPS to initiate TNFα secretion in the presence of TACE inhibitor and assayed for levels of cell surface TNFα staining after 1 hour. Injection of GST alone or of GST-syntaxin2 had no little or no effect on trafficking of TNFα to the cell surface (Fig. 3A). However, GST-

syntaxin4 significantly reduced (> 3 fold) the surface staining of TNFα in the majority of cells.

These data, together with the LPS-responsive expression of syntaxin4, strongly suggest that syntaxin4 plays a crucial role in the docking and fusion of TNFα-containing secretory vesicles at the macrophage cell surface. Cells microinjected with the syntaxin4 fusion protein, but not with either GST alone nor with GST-syntaxin2, were noticeably reduced in size and did not exhibit the dramatic cell ruffling typically induced by activation of macrophages. This further suggests that disrupting the function of syntaxin4 might have a more profound effect on membrane traffic to the cell surface and indeed on the ability of macrophages to undergo morphological changes required for activation and acquisition of their immune-competent phenotype.

Example 3 Experiments were carried out to investigate if syntaxin4 or muncl 8c were a rate-limiting factor for TNFα secretion.

TNFα frafficking in macrophages that were transfected with the full length syntaxin4 cDNA containing a HA-epitope to distinguish it from the endogenous protein was examined. Recombinant HA-syntaxin4 was transported to the cell surface in the same way as the endogenous syntaxin4 protein. Strikingly, overexpression of syntaxin4 in macrophages resulted in a significant increase in cell surface levels of TNFα (Fig. 3C).

These data indicate that overexpression of HA-syntaxin4 facilitated or augmented the delivery of TNFα to the cell surface. Consistent with this a highly

significant correlation between TNFα surface staining and the level of HA- syntaxin4 per cell was observed. Low levels of HA-syntaxin4 expression did not noticeably increase TNFα surface staining, whereas higher (> 2-fold) overexpression of syntaxin4 increased (> 2-fold) cell surface TNFα.

Similar experiments were also performed in cells overexpressing GFP- tagged, full length munclδc (FIG. 3B). Muncl8c-GFP cDNA (10 μg) was introduced into RAW264 cells by electroporation; and 24 hours later cells were treated with LPS to induce synthesis of TNFα and with TACE inhibitor for 1 hour prior to fixation. Unpermeabilized cells were immunostained to detect surface TNFα which was then assessed on GFP-labeled cells compared to surrounding cells. In contrast to cells overexpressing HA-syntaxin4, no significant effect of GFP-muncl8c overexpression on TNFα delivery to the cell surface (Fig. 3B) was observed. There are several models for how SM proteins might participate in membrane fusion. Muncl 8c overexpression failed to affect secretion, either because its role is not rate-limiting or because the high muncl 8c levels induced by LPS already fulfil any requirement for it. Finally, it is possible that overexpressed muncl 8c is not available for events at the plasma membrane since a large proportion of it is found in the cytosol. Example 4

Microarray analysis was performed to provide information on the LPS- induced regulation at the gene level of current drug targets and to identify new drug targets (FIG. 4). Trafficking genes on the microarray were identified according to their

Affymetrix annotation in combination with additional database mining strategies to verify sequence identities. Gene expression levels for each one were recorded after 2 hours and 12 hours of LPS treatment as compared to the controls (untreated). Several sets of genes corresponding to trafficking protein families are shown in FIG 4A which demonstrates the wide-spread changes induced by LPS in general amongst frafficking proteins. The scope and magnitude of gene regulation seen for frafficking proteins in macrophages is novel, in most cells these are largely regarded as unregulated, constitutively-expressed housekeeping genes. This pattern of regulation of trafficking proteins appears to be unique to macrophages undergoing immune activation. The individual responses of genes known to be generally involved in regulating frafficking through the biosynthetic or secretory pathways were then analysed. A subset of these data is shown in FIG. 4B.

The first set of drag targets, syntaxin4, SNAP 23, Muncl 8c, Racl and Cdc42 were selected initially based on their increased or decreased protein levels at 2 hours after LPS treatment at the time of peak TNFα trafficking. We have demonstrated that all of these proteins function in the TNFα pathway and are bona fide drug targets for our anti-TNFα therapeutic strategy. To further understand the basis of the protein up or down regulation by LPS, the changes induced by LPS at the level of gene transcription were investigated. From the microarray analysis described below (FIGS. 4B & 5) we were able to determine whether each of these drug targets is regulated at the gene or protein level by LPS. Syntaxin4, Racl and Cdc42 did not show significant regulation at the gene level in this analysis indication that in each case they are regulated at the protein level only by LPS. Muncl 8c and SNAP23 showed significant upregulation by LPS at the gene level, showing that LPS alters transcription of these proteins. Identification of new drug targets

Microarrays were screened to identify additional trafficking proteins as potential drug targets for regulating TNFα trafficking. Of the many trafficking proteins regulated by LPS, members of the syntaxin and VAMP families of SNARE proteins involved in vesicle docking and fusion were particularly responsive to LPS. The proteins encoded by the LPS-responsive trafficking genes were then assayed by immunoblotting to determine whether changes in gene expression were reflected in equivalent changes at the protein level.

The following criteria were used to select trafficking proteins as potential drug targets: i) Trafficking proteins that are known to operate in secretory pathways showing significant increases or decreases in gene expression and/or protein expression at 2 hours post-LPS concomitant with the onset of peak TNFα secretion. These are deemed likely to regulate TNFα secretion pending further functional assays; and ii) Trafficking genes known to operate in trafficking pathways that are not necessarily LPS responsive but which we have directly implicated in regulating TNFα secretion through functional assays.

Example 5 Validation of the single cell assay for measuring TNFα delivery to the cell surface

The single cell immunofluorescence assay (see FIG. 2) for measuring the trafficking of TNFα (specifically post-golgi frafficking and delivery of TNFα to the cell surface) is potentially a more accurate means of assaying levels of total biologically-active or disease-causing TNFα in macrophage populations or at inflammation sites, because it accounts for both secreted TNFα and surface- retained TNFα. ELISA assays measure only soluble / circulating TNFα and not surface-retained TNFα.

The assay allows measurement of the function of specific regulators in TNFα trafficking in cells where the target molecule has been manipulated with specific drugs, or by overexpressing cDNAs or RNAs, or by microinjecting peptides, cDNAs or antibodies to enhance or block its function.

We have successfully demonstrated the efficacy of the assay for implicating syntaxin4 in cell surface delivery of TNFα in cells overexpressing syntaxin4 and by blocking the function of syntaxin4 with injected peptides (Pagan et al, 2003). The single cell assay will be integrated as part of the drug target discovery pipeline to identify molecules with the capacity to block TNFα secretion.

The assay can also be used as a high-throughput assay to test possible anti- TNFα drags. To demonstrate this application of the assay a number of known drugs, which have previously-established effects on post-golgi trafficking in other cells, have been tested in this assay. The assay was scaled up to a 48 well plate medium-throughput mode using manual fluorescence imaging to collect data and image analysis to quantify the results.

Results from the test experiments using known drugs to block the synthesis or trafficking of TNFα are shown in Table 1. Cells were set up in 48 well plates. Cells were treated with LPS to initiate TNFα synthesis and a TACE inhibitor to block cleavage of the TNFα once it reached the surface of the cell. After 1 hour, cells were fixed, immunostained a first time to label cell surface TNFα, then permeabilized and immunostained a second time to label intracellular TNFα in the golgi complex.

Cells were then imaged by epifluorescence microscopy using appropriate filters. Images were analysed using Adobe Photoshop to measure relative fluorescence intensities at the cell surface and at the golgi complex in individual cells (50 cells per condition).

The results generate several pieces of information. The amount of cell surface fluorescence as a percentage of the total fluorescence in a cell is used as a measure of the TNFα delivery to the cell surface. This value will reveal drags or mediators that primarily affect the post-golgi transport of TNFα (examples are shown in Table 1). Drags that perturb the cytoskeleton reduce the delivery of TNFα to the cell surface by more than 50% while drugs affecting golgi compartments have a range of effects from a 25% reduction, to a total block in transport and in one case even increase transport. These particular drugs are probably too non-specific to be used effectively for the control of TNFα secretion in inflammation. However, their effects demonstrate: i) the efficacy of the assay in measuring TNFα trafficking and the potential for the assay to be used for drag screens; and ii) the efficacy of manipulating elements within the trafficking pathway as a means of blocking or reducing TNFα secretion. Additional data on drug effects can be derived from the same data used to produce Table 1, for instance the total fluorescence (golgi complex plus cell surface) can be used as an indication of the level of overall synthesis of TNFα. This value will reveal drugs or mediators that block, perturb or enhance TNFα synthesis (not shown).

In an alternative embodiment the assay is scaled-up to a 96-well format using fluorimetry and automated image analysis. This can be used as a fully robotic, high-throughput assay which is suitable for large scale testing of thousands of putative drags or small molecule inhibitors. General discussion

Transcriptional and post-translational mechanisms initiated by LPS through surface receptors ensure a rapid and abundant synthesis of TNFα (Raabe et al, 1998, J. Biol. Chem., 273, 974-980). Newly-synthesized TNFα then accumulates in abundance in the golgi complex and is trafficked to the cell surface in small vesicles or tubulo-vesicular carriers for secretion (Shurety et al, 2000; Journal of Interferon and Cytokine Research, 20, 427-438; Shurety et al, 2001, Laboratory Investigation, 81, 107-117). LPS activation therefore creates a sudden requirement for increased exocytotic or secretory cytokine traffic in macrophages. The appropriate trafficking proteins are coordinately regulated to provide the 'machinery' required to accommodate increased membrane traffic and secretion of cytokines. Specific trafficking proteins appear to be regulated in a unique fashion in macrophages, compared to other cell types. Vesicular trafficking of cytokines in macrophages involves a sophisticated and specialized series of processes, which utilize cutomised versions or combinations of ubiquitous cellular machinery. When carrier vesicles are transported to the plasma membrane, they undergo a multi-step process of docking at the membrane via specific protein-protein interactions, followed by membrane-membrane fusion.

The inventors proposed that LPS might induce changes in the trafficking machinery proteins involved in the secretory pathways. Accordingly, the expression of a variety of trafficking proteins at 2 hours (when TNFα secretion peaks) and at 24 hours (after secretion of the early-response cytokines such as TNFα has ceased) was analysed. SNARE proteins, which play a vital role in the docking and fusion of transport vesicle, responded vigorously to LPS-activation and significant increases or decreases in the protein levels were detected.

The possibility that the expression of the t-SNARE components syntaxin4 and/or muncl 8c may be rate-limiting for TNFα secretion was investigated. When macrophages were transfected to overexpress high levels of fully functional syntaxin4, TNFα delivery to the cell membrane was increased compared to cells with normal levels of endogenous syntaxin4. This finding suggested that the amount of TNFα secreted is related directly to the amount of syntaxin4, which in turn correlates with the number of available vesicle docking and fusion sites. The increased level of syntaxin4 and the finding that overexpression of syntaxin4 increases TNFα delivery to the cell membrane demonstrates that the upregulation of t-SNARE proteins that was observed in LPS-treated macrophages is likely to be a necessary in vivo event for the secretion of TNFα. Overexpression of muncl 8c did not alter the delivery of TNFα, probably because it is already significantly upregulated by LPS and is effectively present in excess or is not directly needed for TNFα delivery.

Next, cells were microinjected with an inhibitory peptide, one that essentially mimics the cytoplasmic tail of syntaxin4 and acts as a competitive inhibitor. Cells injected with the syntaxin4 peptide, but not with a syntaxin2 peptide, showed dramatically reduced delivery of TNFα to the cell surface compared to surrounding cells. Thus syntaxin4 is required for surface delivery of TNFα and effectively reducing the number of t-SNAREs blocked trafficking of TNFα. Therefore modulating the levels of syntaxin4 can have a profound modulatory effect on macrophage function, by inhibiting its ability to secrete TNFα. We have identified syntaxin4 as an essential regulator of TNFα secretion and as a potential drug target for anti-TNFα therapies.

Drags designed to target syntaxin4 and other key proteins of the molecular machinery controlling the transport, docking, membrane fusion and exocytosis of vesicles in macrophages would cripple the macrophage' s ability to secrete cytokines and to respond to an immune challenge. Genetic manipulation, for example silencing a specific, key trafficking protein at the post-transcriptional level would similarly affect TNFα secretion. The LPS-treated macrophages were also screened to identify trafficking proteins involved in cytokine secretion based on their responses to LPS at the level of gene transcription. Microarray analyses demonstrated even more dramatically than studies at the protein level, that LPS regulates many trafficking proteins. The inventors identified additional possible regulators of TNFα secretion in this manner, based on their significant up or down regulation coincident with peak TNFα frafficking. Additional members of SNARE complexes, syntaxins 3 and 6, Vtil-B and VAMP3 have been identified as well as other proteins, VapB and RGS 16. The microarray analysis also demonstrated that syntaxin4, Racl and Cdc42 did not show significant regulation at the gene level but are regulated at the protein level only by LPS, presumably through post- transcriptional mechanisms. Muncl 8c and SNAP23 showed significant upregulation by LPS at the gene level, showing that LPS alters transcription of these proteins. SNARE proteins VAMP3, syntaxin3, syntaxinδ and Vtil-B were upgraded at both the gene and protein levels. RGSGAIP was upregulated at the protein level.

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference. Table 1

Claims

CLAIMS 1. A method of regulating an activity of a pro-inflammatory cytokine including the step of modulating secretion of said cytokine by a cell or tissue of an animal by modulating one or more intracellular frafficking proteins.

2. The method of claim 1 wherein the activity of the pro-inflammatory cytokine is regulated in vivo.

3. The method of claim 1 wherein the activity of the pro-inflammatory cytokine is regulated in vitro.

A. The method of claim 1 wherein secretion of the pro-inflammatory cytokine is modulated to thereby modulate an immune response of said animal.

5. The method of claim 1 wherein said pro-inflammatory cytokine is selected from the group consisting of TNFα, interleukin-6 and interleukin-10.

6. The method of claim 5 wherein said pro-inflammatory cytokine is TNFα.

7. The method of claim 1 wherein one or more intracellular frafficking protein regulates post-golgi transport of said cytokine.

8. The method of claim 7 wherein said intracellular trafficking protein(s) are selected from the group consisting of syntaxin4, RGS 16, RGSGAIP, RGS, synatxin3, syntaxinό, Vtil-B, Muncl 8c, Cdc42, VapB and Rac-1.

9. The method of claim 1 wherein secretion of the pro-inflammatory cytokine is inhibited, suppressed or otherwise reduced during an immune response in the animal in response to an acute or chronic inflammatory disease.

10. The method of claim 9 wherein said acute or chronic inflammatory disease is selected from the group consisting of septic shock, inflammatory bowel disease, arthritis, psoriasis, congestive heart disease and chronic pulmonary disease.

11. The method of claim 9 wherein said immune response of the animal is primed, augmented or otherwise improved in response to infection.

12. The method of claim 1 wherein said cell is a macrophage.

13. The method of claim 1 wherein said animal is a mammal.

14. The method of claim 13 wherein said mammal is a human.

15. The method of claim 1 wherein modulating secretion of said pro- inflammatory cytokine is by an inhibitor of pro-inflammatory cytokine secretion.

16. The method of claim 15 wherein said inhibitor of pro-inflammatory cytokine secretion targets syntaxin4 and/or muncl 8c.

17. The method of claim 15 wherein said pro-inflammatory cytokine is selected from the group consisting of TNFα, interleukin-6 and interleukin-10.

18. The method of claim 17 wherein said pro-inflammatory cytokine is TNFα.

19. A pharmaceutical composition comprising a modulator of pro-inflammatory cytokine secretion which modulates intracellular trafficking of said cytokine.

20. The pharmaceutical composition of claim 19 wherein said modulator is selected from the group consisting of an antibody, peptide, nucleic acid, drug and mimetic.

21. The pharmaceutical composition of claim 19 wherein said modulator targets at least one protein selected from the group consisting of syntaxin4, RGS 16, RGSGAIP, RGS, synatxin3, syntaxinό, Vtil-B, Muncl 8c, Cdc42, VapB and Rac- 1.

22. The pharmaceutical composition of claim 19 wherein said modulator is an inhibitor of pro-inflammatory cytokine secretion that targets syntaxin4 and/or Muncl 8c.

23. A method of identifying one or more intracellular frafficking proteins that regulate pro-inflammatory cytokine secretion, said method including the step of identifying one or more proteins that colocalise with said cytokine in a cell, thereby indicating that at least one of said one or more proteins is an intracellular frafficking protein that regulates secretion of said cytokine.

24. A method of identifying one or more intracellular trafficking proteins that regulate cytokine secretion including the step of determining whether expression of the one or more intracellular frafficking proteins or nucleic acid encoding same changes in response to stimulation or activation of cytokine production by a cell or tissue, thereby indicating that at least one of said one or more proteins is an intracellular trafficking protein that regulates secretion of said cytokine.

25. The method of claim 24 wherein the expression of the intracellular trafficking protein or nucleic acid increases in response to stimulation or activation of cytokine production by a cell or tissue.

26. The method of claim 24 wherein the expression of the intracellular frafficking protein or nucleic acid decreases in response to stimulation or activation of cytokine production by a cell or tissue.

27. The method of claim 24 wherein expression of the intracellular frafficking protein or nucleic acid encoding same is detected by a microarray technique.

28. The method of claim 24 wherein identification of the intracellular frafficking protein or nucleic acid is detected by a protein array technique.

29. The method of claim 24 wherein said intracellular trafficking protein is detected by immunoblot.

30. A method of identifying a modulator of pro-inflammatory cytokine secretion, said method including the steps of:

(i) administering a candidate modulator to a cell that secretes a pro- inflammatory cytokine; and (ii) determining whether said candidate modulator affects secretion of said cytokine by said cell.

31. The method of any one of claim 23, claim 24 or claim 30 wherein said cell is a macrophage.

32. The method of claims 23, 24 or 30 wherein said pro-inflammatory cytokine is selected from the group consisting of TNFα, interleukin-6 and interleukin-10.

33. The method of claim 32 wherein said pro-inflammatory cytokine is TNFα.

34. The use of an intracellular trafficking protein for identification, production or design of a modulator of pro-inflammatory cytokine secretion.

35. The use of the intracellular frafficking protein of claim 34 wherein the infracellular trafficking protein is selected from the group consisting of syntaxin4, RGS16, RGSGAIP, RGS, synatxin3, syntaxinό, Vtil-B, Muncl8c, Cdc42, VapB and Rac-1.

36. The use of the intracellular trafficking protein of claim 34 wherein the pro- inflammatory cytokine is selected from the group consisting of TNFα, mterleukin-6 and interleukin-10.