WO2013134292A2 - Compositions and methods for mitigation of inflammasome activation - Google Patents

Description

Title: COMPOSITIONS AND METHODS FOR MITIGATION OF

INFLAMMASOME ACTIVATION

Cross-Reference to Related Application:

[0001] This application claims priority from U.S. Provisional Application No. 61/608,137, entitled "COMPOSITIONS AND METHODS FOR MITIGATION OF INFLAMMASOME ACTIVATION" filed March 8, 2012, the disclosure of which is incorporated by reference herein.

Background of the Invention

[0002] This invention relates generally to compositions and methods for treating an infection, methods for modulating an immune response, methods for preventing disease, methods for preventing an infection, or combinations of these such as methods of treating a disease and preventing a secondary infection using agents and compositions of the invention, In particular, the invention relates to compositions and methods of treating inflammation such as, but not limited to, systemic inflammation, chronic inflammatory diseases, and inflammation due to sepsis, non-septic injury, trauma, surgery or combinations thereof by administering the agents provided by embodiments of the invention. Embodiments of the invention may be administered to a subject by any method known in the art including, but not limited to, enteral, parenteral, and topical delivery.

[0003] In the development of the present invention a strategy has been employed of modulating the host tolerance/resistance mechanisms as a means to ablate infection from progressing to disease. Mammals have evolved tolerance mechanisms to accommodate beneficial microorganisms as well as potential pathogens and have evolved resistance mechanisms to reduce the pathogen burden during infection. These two types of mechanisms have been reviewed by Schneider and Ayers¹ and they classify the regulators of tolerance and resistance into three basic classes. In class one mechanisms, resistance and tolerance are linked

¹ Schneider, D. S. & Ayers, J. S. Two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases. Nat. Rev. Immunol. 8:889-895 (2008). and their effects are opposite. Reactive oxygen species are an example of this class. The second class is comprised of regulators that control both resistance and tolerance in an either/or manner and are therefore separable as opposed to an absolute linkage as in the class one effector molecules. The third class is regulators that effect only tolerance. The inflammasome signaling platforms represent class two and class three regulators.

[0004] The inflammasome comprises a family of cytosolic receptors called NOD -like receptors (NLR) that are involved in innate immune recognition of pathogen associated molecular patterns as well as intracellular and extracellular damage associated molecular patterns². Thus far, more than 20 inflammasomes have been identified and many are present in nearly every cell type. NLRP3 is the most extensively studied inflammasome and has been found to be activated by a diverse range of stimuli including microbial derived products³'⁴'⁵'⁶,

3 7 8 9 10 11 12 13

environmental factors ' ' ' and endogenous molecules ' ' ' . The NLRP3 inflammasome would represent a class two regulator of resistance/tolerance. The NLRP6 inflammasome, on the

² Lamkanfi, M. Emerging inflammasome effector mechanisms. Nat. Rev. Immunol. 11:213-220 (2011).

Dostert, C, et al. Malarial hemozoin is a Nalp3 inflammasome activating danger signal. PLoS One. 4:e6510 (2009)

⁴ Gurcel, L., et al. Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival. Cell. 126: 1135-45 (2006)

⁵ Chu, J., et al. Cholesterol-dependent cytolysins induce rapid release of mature IL-lbeta from murine macrophages in a NLRP3 inflammasome and cathepsin B -dependent manner. J. Leukoc. Biol. 86: 1227-38 (2009)

⁶ Thomas, P. G., et al. The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1. Immunity 30:566-75 (2009).

⁷ Hornung, V., et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9:847-56 (2008).

⁸ Cassel, S. L., et al. The NALP3 inflammasome is essential for the development of silicosis. Proc. Natl. Acad. Set, USA 105:9035-40 (2008).

⁹ Dostert C, et al. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 320:674-7 (2008).

¹⁰ Yamasaki, K., et al. NLRP3/cryopyrin is necessary for interleukin-lbeta (IL- lbeta) release in response to hyaluronan, an endogenous trigger of inflammation in response to injury. J Biol Chem. 284: 12762-71 (2009).

¹¹ Salminen, A., et al. Amyloid-beta oligimers set fire to inflammasomes and induce Alzheimer's pathology. J Cell Mol Med. 12:2255-62 (2008).

¹² Mariathasan, S., et al. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature. 440:228-32 (2006).

¹ Gasse, P., et al. Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis. Am JRespir Crit Care Med. 179:903-13 (2009). other hand, has been recently shown to regulate the composition of the microbiota in the colon and therefore would represent a class three regulator of tolerance¹⁴.

[0005] Previously, we described a new molecular entity, acALY18, and the surprising discovery that its regulation of the NLRP3 inflammasome is a safe and effective means to prevent Gram-positive and Gram-negative bacterial infections from progressing to disease (enhanced tolerance) as well as abrogate disease progression in an established infections (enhanced resistance). Furthermore, we describe acALY18 regulation of the inflammasome as an effective therapeutic means to clear viral infections and intracellular bacterial infections. Surprisingly, a single pharmaceutical agent can be effective both as a prophylactic and a therapeutic against bacterial and viral pathogens while selective pressure for development of pathogen resistance is greatly reduced or altogether absent.

[0006] Herein, we describe the induction of acALY18 in response to cellular stress and the intracellular protein target for acALY18 that is up-stream of the inflammasome assembly and activation.

[0007] U.S. Patent No. 7,608,589 B2, PCT Application Nos. US2009/04159, and other publications are used herein for reference to illuminate the background of the invention or provide additional details regarding the biological mechanism of action are incorporated by reference.

Summary of the Invention

[0008] Embodiments of the invention described herein include compounds comprised of a peptide of about 5 to about 25 amino acids that binds to one or both iso forms of X Box Protein 1 ("XBPls" or "XBPlu"). The peptide of the embodiments of the invention may include a peptide or peptide fragment with the amino acid sequence XLYDKGYTSKEQKDCVGIX or XLYDKGYTPKEQKDCVGIX (collectively or individually "acALY18") or XLYDKGYTSKEQKDCVGX or XLYDKGYTPKEQKDCVGX (collectively or individually

Elinav, E., et al. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell 145:745- 757 (2011). "acALY17") or inversions or mimetics thereof, wherein X is absent or a naturally occurring amino acid or mimetic thereof, a derivatized amino acid or a non-amino acid prosthetic group.

[0009] In certain embodiments of the invention, acALY18 or acALY17 binds to XBPl in a molar ratio of 1 : 1 , or 2 : 1 , or 3 : 1 but most preferably in a molar ratio of 3 : 1.

[0010] In certain embodiments of the invention, acALY18 or acALY17 binds one or more of the cysteine amino acids at positions 204, 215 and 247 in XBPlu to form one or more disulfide bonds that covalently link acALY18 or acALY17 to XBPlu.

[0011] In certain embodiments of the invention binding of acALY18 or acALY17 with XBPl induces assembly and activation of one or more of the intracellular inflammasomes and most preferably the NLRP3 inflammasome to induce an immune response and more specifically an innate immune response in an animal.

[0012] The immune response, in certain embodiments of the invention, may include inducing an immune response for at least 1 -3 days following administration of the agent, and in certain other embodiments, the immune response may be induced for a week or more.

[0013] In still other embodiments of the invention, the agent may be deposited in tissue of the subject, and the agent may be administered by any method including enteral, parenteral, and topical delivery or combinations of these. Parenteral administration may include, but not limited to, intra-articular, intrasynovial, intrathecal, intraarterial, intravenous, intramuscular, subcutaneous or combinations thereof. Enteral administration may include, but not limited to, oral, peroral, rectal, vaginal or combinations thereof, and topical administration may include, but not limited to, intranasal, intrarespiratory, epicutaneous, transdermal delivery or combinations thereof.

[0014] In some embodiments of the invention, the agent may be administered prior to exposure to a disease causing agent and may, in these embodiments, substantially prevent disease onset or progression. The agent may also be delivered following exposure to a disease causing agent and may, in these embodiments, substantially arrest disease progression. In yet further embodiments, the agent may be delivered to the subject following disease onset to lessen the severity of the disease or reverse the course of the disease. [0015] Further methods of embodiments of the invention include methods for treating an infection, methods for modulating an immune response, methods for preventing disease, methods for preventing an infection, or combinations of these such as methods of treating a disease and preventing a secondary infection using agents and compositions of the invention described hereinabove.

[0016] Embodiments of the invention further include the surprising discovery that acALY18 or acALY17 binding to XBP1 can inhibit infiammasome dependent expression of collagen in fibrotic diseases like systemic scleroderma, chronic obstructive pulmonary disease, or cystic fibrosis.

[0017] Embodiments of the invention further include methods of treating inflammation such as, but not limited to, systemic inflammation, chronic inflammatory diseases, and inflammation due to sepsis, non-septic injury, trauma, surgery or combinations thereof by administering the agents of embodiments of the invention. Embodiments of the invention may be administered to a subject by any method known in the art including, but not limited to, enteral, parenteral, and topical delivery.

[0018] For a fuller understanding of the nature and advantages of the present invention, reference should be made to the following detailed description taken in connection with the accompanying drawings.

Brief Description of the Drawings

[0019] FIG. 1. acALY18 localizes to the cytosol and does not induce ER stress as measured by GRP78 expression. Primary human fibroblasts treated with 50 nM acALY- AlexaFluor594 showing intracellular distribution (above left) and acALY18 detected on Western as a trimer at 6 kDa (inset, arrow). (A) Western analysis of hu-fibroblasts treated with 25 nM Thapsigargin and probed for acALY18. (B) Western analysis of hu-fibroblasts treated with Thapsigargin, acALY18, mock treatment, and no treatment for 24 h. (C) Time course for Thapsigargin induced GRP78 expression. [0020] FIG. 2. acALY18 is derived from TRPC1. Western analysis of wild-type (WT) embryonic mouse fibroblasts (A) and TRPC1^"7" embryonic mouse fibroblasts (B) after treatment with 25 nM Thapsigargin and probed for acALY18. (C) acALY18 expression in WT and TRPC ^7" embryonic mouse fibroblasts normalized to GAPDH. (D) IL-Ιβ expression from WT and TRPC ^7" fibroblasts treated with Thapsigargin for 72 h.

[0021] FIG 3. acALY18 is co-expressed with XPBlu. Western analysis of hu-fibroblasts treated with 25 nM Thapsigargin and probed for acALY18 (panel A) or XBP1 (panel B) at various time points post-treatment. (C) Expression of acALY18 and (D) XBPlu normalized to GAPDH.

[0022] FIG 4. acALYl 8 forms a disulfide bond with its protein target. (A) GM04190 hu-fibroblasts were treated for 30 min. with 25 nM Thapsigargin (Tg). The whole cell lysates were reduced with DTT/BME for 15, 30, or 60 min. and analyzed by Western. (B) acALYl 8 levels were normalized to GAPDH.

[0023] FIG 5. Pull-down assay with XBP1 antibody after 1 h and 24 h treatment with 25 nM Thapsigargin. Primary hu-fibroblasts were treated with 25 nM Thapsigargin for 1 h and 24 h. Cell lysates incubated with XBPl antibody immobilized on Protein A coated magnetic beads. The bead eluate was analyzed by Western and probed for XBPl (left) and acALYl 8 (right). Densitometry analysis (bottom panel) indicates increased expression of acALYl 8 associated with XBPl consistent with the whole cell lysate Western analysis shown in FIG. 3.

[0024] FIG 6. Immunoprecipitation of XBPl/acALY18 complex. Immunoprecipitated protein isolated with acALYl 8 antibody (right) or XBPl antibody (left).

[0025] FIG. 7. Amino acid sequence of XBPlu and showing the putative binding domain for acALY18. The complete amino acid sequence of the unspliced isoform of XBPl (XBPlu) is depicted (Top). The Nuclear Exclusion Sequence domain (AAs 185-208) is highlighted in pink and the putative proteolytic degradation sequence domain (AAs 209-261) is highlighted in blue. (Bottom) the putative acALYl 8 binding domain (AAs 201 -220) associated with 2 molecules of acALYl 8 showing the perfect alignment of cysteines to form the corresponding disulfide bond. [0026] FIG. 8. Comparison of XBP1 mRNA splicing induced by Thapsigargin or acALY18. Southern blot analysis of XBP1 mRNA expression in murine fibroblasts treated with either 3 ng/ml of acALY18 or 25 nM Thapsigargin.

[0027] FIG. 9 Comparison of the effects of LPS and acALY18. Primary hu-fibroblasts were treated with either 5 ng/mL acALY18 or 100 ng/mL LPS for 1 , 4, or 24 h and the expression of XBPlu was measured by Western (Top). XBPlu expression declined over time in the acALY18 treated cells but increased in the LPS treated cells (Bottom).

Detailed Description of the Invention

[0028] It must also be noted that as used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0029] An "adjuvant" refers to any substance which enhances the immune -stimulating properties of an antigen or the pharmacological effect of a drug.

[0030] As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55.

[0031] The terms "mimetic," "peptide mimetic" and "peptidomimetic" are used interchangeably herein, and generally refer to a peptide, partial peptide or non -peptide molecule that mimics the tertiary binding structure or activity of a selected native peptide or protein functional domain (e.g., binding motif or active site). These peptide mimetics include recombinantly or chemically modified peptides, as well as non-peptide agents such as small molecule drug mimetics, as further described below.

[0032] As used herein, the term "pharmaceutically acceptable salts, esters, amides, and prodrugs" refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.

[0033] As used herein, the term "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration upon a mammal without or with minimal production of undesirable physiological effects such as nausea, dizziness, rash, or gastric upset. In a preferred embodiment, the therapeutic composition is not antigenic when administered to a human patient or other animal for therapeutic purposes.

[0034] "Providing" when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted.

[0035] As used herein "subject" or "patient" refers to an animal or mammal including, but not limited to, human, dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rabbit, rat, mouse, etc.

[0036] "Disease" for purposes of the present invention may be any infectious agent such as, for example, viral particles, bacterial pathogens, protozoan parasites, fungal pathogens, metastatic carcinoma cells, and the like. "Diseased" as used in reference to a "diseased subject" may refer to any human or animal subject infected with a disease causing agent. The "diseased subject" may or may not exhibit signs of disease such as, for example, known symptoms.

[0037] As used herein a "sample" includes a biological sample which can be tested by the methods of the present invention and include, but are not limited to, body fluids such as serum, plasma, whole blood, cerebrospinal fluid, lymph fluids, various external secretions (urine, respiratory, intestinal or genitourinary tract secretions, tears, etc.), etc.

[0038] As used herein, the term "therapeutic" means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient. Embodiments of the present invention are directed to stimulate the innate immune response or modulation of the inflammatory response. The methods herein for use contemplate prophylactic use as well as curative use in therapy of an existing condition.

[0039] The terms "therapeutically effective" or "effective", as used herein, may be used interchangeably and refer to an amount of a therapeutic composition embodiments of the present invention~e.g. one or more of the peptides or mimetics thereof. For example, a therapeutically effective amount of a composition comprising acALY18 or acALY17, or mimetics thereof, is a predetermined amount calculated to achieve the desired effect, i.e., to effectively stimulate (or down-regulate as appropriate) an innate immune response in an animal to whom the composition is administered.

[0040] The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., excipient, carrier, or vehicle.

[0041] One embodiment of the present invention may be directed to acALY18. Other embodiments of the invention may include compositions containing acALY18, compositions that contain portions of acALY18, compositions containing analogs of acALY18 and compositions containing peptide mimetics of acALY18.

[0042] In embodiments of the invention, acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may be provided to a subject and may stimulate therapeutic effects such as, but not limited to, inducing an immune response, and in certain embodiments, the immune response may be an innate immune response in the subject so provided. acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18, in other embodiments of the invention, may be administered to a subject undergoing treatment for disease, to a subject that is healthy, or to a subject that is healthy and may be exposed to disease, disease forming agents, or diseased humans and/or animals. In embodiments of the invention where acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18, and therapeutics containing acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 are provided to a subject that is healthy, the acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may promote the prophylactic activation of an immune response of the subject, and in certain embodiments prophylactic activation of innate immunity.

[0043] Without wishing to be bound by theory, the acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18, in embodiments of the invention, when provided to a subject may activate tissue resident immune cells in the subject. In some embodiments, the acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may initiate an immune response, and in other embodiments, the immune response may be an innate immune response.

[0044] Embodiments of the present invention may also include methods of administering acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 and therapeutics containing acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 such as, but not limited to, parenteral, enteral, or topical administration.

[0045] Other embodiments of the present invention include antibodies with specificity for acALY18, and methods for the use of such antibodies in depleting systemic or localized concentrations of acALY18 or acALY18 containing proteins in a subject. In certain embodiments, the subject provided with antibodies specific to acALY18 may exhibit symptoms of immune disease such as, but not limited to, systemic inflammation, chronic inflammatory diseases, atherosclerotic disease, rheumatoid diseases, autoimmune diseases, and the like.

[0046] Still other embodiments of the invention include fluorescently labeled acALY18, analogs of acALY18, and fluorescently labeled antibodies or antibody fragments with specificity to acALY18 and methods for producing such fluorescently labeled acALY18, analogs of acALY18 and antibodies. Fluorescently labeled acALY18, analogs of acALY18 and antibodies may be used, in embodiments of the invention, as diagnostic tools to assess aspects of the immune system and immunopathology both in vitro and in vivo, and in some embodiments, subjects may include, but not limited to, subject exhibiting symptoms consistent with chronic inflammatory disease, autoimmune disease, atherosclerotic disease, diabetes and the like.

[0047] acALY18 described in embodiments of the invention may be of general formula (I) or (II):

XLYDKGYTSKEQKDCVGIX

(I)

XLYDKGYTPKEQKDCVGIX

(Π)

[0048] acALY17 described in embodiments of the invention may be of general formula (III) or (IV):

XLYDKGYTSKEQKDCVGX

(III)

XLYDKGYTPKEQKDCVGX

(IV)

[0049] In such embodiments, X may be absent or any naturally occurring amino acid or mimetic thereof, derivatized amino acids or non-amino acid prosthetic groups, and in particular embodiments, the N-terminal most amino acid may be N-acetyl alanine (acA). Without wishing to be bound by theory, the peptide sequences I and II presented herein above may be representative of a larger class of peptides which may trigger an immune response in a mammal and, in particular, a human when administered alone or conjugated with a lipid moiety, for example, diacylglycerol. Thus, any peptide isolated from virtually any source that performs such a function may be encompassed by embodiments of the invention including, for example, peptides associated with lipids or proteins or carbohydrates isolated from sources such as, but not limited to, animals, mammals, humans, primates, cows, horses, pigs, birds, reptiles, insects, microorganisms, bacteria, and so on. [0050] Additionally, in such embodiments, X may be absent or any naturally occurring amino acid or mimetic thereof, derivatized amino acids or non -amino acid prosthetic groups, and in particular embodiments, the N-terminal most amino acid may be N-acetyl alanine (acA). Without wishing to be bound by theory, the peptide sequences III and IV presented herein above may be representative of a larger class of peptides which may down-regulate an immune response in a mammal and, in particular, a human when administered alone or conjugated with a lipid moiety, for example, diacylglycerol. Thus, any peptide isolated from virtually any source that performs such a function may be encompassed by embodiments of the invention including, for example, peptides associated with lipids or proteins or carbohydrates isolated from sources such as, but not limited to, animals, mammals, humans, primates, cows, horses, pigs, birds, reptiles, insects, microorganisms, bacteria, and so on.

[0051] The peptide may be covalently conjugated to the lipid moiety by esterification at the carboxyl-terminal carboxylic acid of the peptide, and in some embodiments, the peptide moiety may be conjugated to the lipid moiety through a phosphoester at the carboxyl-terminal carboxylic acid of the peptide.

[0052] The sequence of the peptide may vary among embodiments. For example, in one embodiment the peptide sequence may be any of compounds I-IV or synthetic equivalents, peptide analogs or peptidomimetics thereof.

[0053] A variety of techniques are available for constructing peptide mimetics with the same or similar desired biological activity as the corresponding native peptide but with better solubility, stability, and/or susceptibility to hydrolysis or proteolysis. Therefore, these characteristics of peptidomimetic compounds encourage their use in therapeutic applications since they may have increased cell permeability, greater affinity and/or avidity for cell receptors and prolonged biological half-life. Certain peptidomimetic compounds are based upon the amino acid sequence of the peptides of the invention. Often, peptidomimetic compounds are synthetic compounds having a three-dimensional structure (i.e., a "peptide motif) based upon the three-dimensional structure of a selected peptide. The peptide motif provides the peptidomimetic compounds with the desired biological activity, i.e., enhancing or stimulating an immune response, wherein the binding activity of the mimetic compound is not substantially reduced, and is often the same as or greater than the activity of the native peptide on which the mimetic is modeled.

[0054] Peptidomimetic design strategies are readily available in the art. One class of peptidomimetics contains a backbone that is partially or completely a non -peptide, but mimics the peptide backbone atom-for atom and comprises side groups that likewise mimic the functionality of the side groups of the native amino acid residues. Several types of chemical bonds, such as ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics. Another class of peptidomimetics is a small non-peptide molecule that binds to another peptide or protein, but which is not necessarily a structural mimetic of the native peptide. Yet another class of peptidomimetics has arisen from combinatorial chemistry and the generation of massive chemical libraries. These generally are novel templates which, though structurally unrelated to the native peptide, possess necessary functional groups positioned on a non-peptide scaffold to serve as "topographical"' mimetics of the original peptide.

[0055] Hereinafter, the terms acALY18 and acALY17 may be used interchangeably and are taken to mean any of the chemical structures I-IV, portions of structures I-IV, analogs, or mimetics without regard to theory or convention.

[0056] Without wishing to be bound by theory, acALY18, portions of acALY18, analogs of acALY18, or mimetics of acALY18 may be activated within target tissues producing the activated acALY18 ("acALY18"). The "acALY18" moiety may then bind to XBPlu in or on immune cells and/or non-immune cells as previously noted and initiate the release of cytokines such as, but not limited to IL-Ιβ, IL-18, IL-33 IL-6, IL-8, MCP-1, ΜΙΡ-Ια and β, INF-γ, TNF-α, Granzyme, RA TES, and Caspase-1 recruiting and activating monocytes, macrophages, dendritic cells, T-cells, phagocytic NK cells, or neutrophils and stimulating the release of other stimulatory cytokines, chemokines, peptides, and proteins. Cytokine release may also stimulate CD5⁺ B-cells (also known as tissue resident Bl cells) to produce immunoglobulin (IgM, a potent opsonizing immunoglobulin) and other B-cell derived immunoglobulins such as IgA or IgG, cytokines and chemokines. Therefore, embodiments of the present invention include acALY18, portions of acALY18, analogs of acALY18, and peptide mimetics of acALY18 that when administered to a subject may be present in a target tissue in an inactive form (i.e. as a portion of a protein) and activated by the action of proteolytic enzymes over time thereby allowing for the maintenance of increased acALY18 concentrations and immune activation in the target tissue of the subject over time. The sustained release of active acALY18 may allow for sustained innate immune activation thereby conferring the prophylactically treated subject with an enhanced ability to fight disease when immunologically challenged over time and therapeutically treated with a long-acting formulation of the acALY18.

[0057] In further embodiments of the present invention, more than one acALY18 peptide may be covalently conjugated or passively adsorbed onto a carrier molecule or particle. Without wishing to be bound by theory, the duration of the effective release of acALY18 peptide, or peptide mimetics, may be directly related to the number of acALY18 peptides, or peptide mimetics, conjugated or adsorbed to the carrier. Therefore, administration of acALY18, portions of acALY18, analogs of acALY18, or mimetics of acALY18 having multiple acALY18 peptide moieties conjugated or adsorbed may release acALY18 peptide over a longer period of time than a similarly administered acALY18, portions of acALY18, analogs of acALY18, or mimetics of acALY18.

[0058] In some embodiments, acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may be delivered directly to a subject, and in others, acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may be combined with a pharmaceutically-acceptable carrier to make a pharmaceutical composition that may be delivered or provided to a subject.

[0059] A variety of administration routes are available in embodiments of the invention. The particular mode selected will depend upon the particular therapeutic effect desired, the severity of the condition being treated, and the dosage required for therapeutic efficacy. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include, but are not limited to, oral, rectal, topical, nasal, intradermal, inhalation, intra -peritoneal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous, subcutaneous, or intramuscular routes are particularly suitable for purposes of the present invention.

[0060] Pharmaceutical compositions of embodiments of the invention may include buffering agents such as, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, phosphoric acid in a salt and the like and, optionally, preservatives, such as: benzalkonium chloride, chlorobutanol, parabens, thimerosal and the like.

[0061] Pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods may include the step of bringing the active agent into association with a carrier that constitutes one or more accessory ingredients. In general, the compositions may be prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0062] Various other carrier materials may also advantageously be present in a nasal spray in appropriate quantities. The solution may be made mildly saline, by dissolving a small amount of sodium chloride in the aqueous medium. The salt concentration may be in the range of about 0.1-2.0% and will preferably be on the order of about 0.65%. Other materials such as surfactants, vitamins and vitamin derivatives, antihistamines, wetting agents, preservatives, moisturizers, emulsifiers, odorants and the like may also be present in conventional concentrations. Numerous disclosures of suitable materials may be found in the literature, along with descriptions of efficacious concentrations in aqueous media. Those skilled in the art will have no difficulty in determining suitable materials and concentrations for their known functions. Delivery of the spray to the nasal cavity may be by any conventional spray technique or device.

[0063] Embodiments of the invention also provide compositions suitable for parenteral administration wherein a sterile aqueous preparation of acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non -toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, and the like administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA which is hereby incorporated by reference in its entirety.

[0064] Delivery systems of embodiments of the invention may be designed to include time- released, delayed release or sustained release delivery systems. acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may also be used in conjunction with additional immunostimulatory or immunoenhancing agents. Using such systems, repeated administrations of acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may be avoided increasing convenience to the subject, and may be particularly suitable for certain compositions of the present invention.

[0065] acALY18, portions of acALY18, analogs of acALY18, mimetics of acALY18 and pharmaceutical compositions including acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may be administered in an effective amount that enhances or stimulates an immune response, and in certain embodiments, in an effective amount to stimulate an innate immune response.

[0066] In general, routine experimentation in clinical trials may be used to determine specific ranges for optimal effect for each agent or pharmaceutical composition and administrative protocol. Administration of acALY18, portions of acALY18, analogs of acALY18, mimetics of acALY18 and pharmaceutical compositions including acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 to specific subjects may be adjusted to within effective and safe ranges depending on the subject's condition and responsiveness to initial administrations. However, the ultimate administration protocol may be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the subject, the potency of the acALY18, portions of acALY18, analogs of acALY18, mimetics of acALY18 and pharmaceutical compositions including acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18, the duration of the treatment and the severity of the disease being treated.

[0067] In embodiments of the invention, a dosage regimen of acALY18, portions of acALY18, analogs of acALY18, mimetics of acALY18 and pharmaceutical compositions including acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may be administered by nasal spray or an inhaler. For nasal spray or inhaler formulations, the ground particle size for effective dissolution or dispersion of the acALY18, portions of acALY18, analogs of acALY18, mimetics of acALY18 and pharmaceutical compositions including acALY18, portions of acALY18, analogs of acALY18 and mimetics of acALY18 may be on the order of about 0.1 to about 20 microns, about 0.2 to about 10 microns, and in certain embodiments, about 0.2 to about 5 microns. Incorporation of acALY18, portions of acALY18, analogs of acALY18, or mimetics of acALY18 into an aqueous carrier may be aided by first dispersing the acALY18, portions of acALY18, analogs of acALY18, or mimetics of acALY18 in a solution such as, for example, a 4% concentration in a lactone solution. Once thoroughly mixed, dispersed, and/or dissolved, acALY18, portions of acALY18, analogs of acALY18, or mimetics of acALY18 may be present at a concentration of from about 0.001 % to about 2.0 %, about 0.01 % to about 0.35 %, and in certain embodiments, about 0.10 %. (All percentages herein are by weight unless otherwise noted.)

[0068] In other embodiments, acALY18, portions of acALY18, analogs of acALY18, or mimetics of acALY18 may be administered orally to achieve total blood levels in the range of from about 2 mg to about 100 mg/day in from two to four divided doses. In some embodiments, intermittent therapy (e.g., one week out of three weeks or three out of four weeks) may be used. [0069] In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that the patient tolerance permits. Multiple doses per day may be used to achieve appropriate systemic effects. Generally, a maximum dose may be used. A maximum dose may be considered the highest safe dose according to sound medical judgment. Those of ordinary skill in the art will understand, however, that a subject may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reason.

[0070] In other embodiments of the invention, at least one acALY18, portion of acALY18, analog of acALY18, or mimetic of acALY18 may be covalently attached to an antigenic peptide or simply mixed with the antigenic peptide or vaccine prior to administration of the antigenic peptide or vaccine to a subject. Without wishing to be bound by theory, the addition of acALY18 may enhance the immunogenicity of the antigenic peptide or vaccine by stimulating the innate immune system at the time of administration of the antigenic peptide or vaccine. Synthetic antigens having covalently attached one or more acALY18 or acALY 18 -antigen or vaccine admixtures may be administered to a subject to induce a long-term adaptive immune response in the subject.

[0071] In further embodiments of the invention, antibodies may be raised to naturally occurring acALY18 and acALY18 peptides, and in still further embodiments, antibodies so raised may be administered to a subject to deplete the concentration of the acALY18 to which the antibody was raised. Without wishing to be bound by theory, administration of acALY18 and/or acALY18 peptide depleting antibodies may be a beneficial therapeutic strategy for subjects exhibiting uncontrolled systemic inflammation such as, for example, sepsis, atherosclerosis, rheumatoid diseases, autoimmune diseases, inflammatory bowel disease, Type I diabetes and the like. In similar embodiments, acALY18 and acALY18 peptide depleting antibodies may be used to treat non-septic injury such as, for example, trauma, inflammation due to extensive surgical procedures and the like.

[0072] Antibodies to acALY18 and acALY18 peptides, of embodiments of the invention, may be raised in rabbits, mice, goats, horses, or other species by methods well known to those skilled in the art. For example, monoclonal antibodies to acALY18 or acALY18 peptides may be raised utilizing the hybridoma fusion techniques, and selected hybridomas may be maintained in cell culture or in a bioreactor for the continuous production of monoclonal antibodies. In some embodiments, the acALY18 peptide specific binding region of a monoclonal antibody may be selectively produced by specific chemical cleavage of the whole antibody or recombinant methods known in the art. In other embodiments, the specific acALY18 binding region may be conjugated to the Fc region of a human antibody to produce a humanized chimera for administration of a acALY18 depleting antibody to human subjects. Chimeric antibodies are well known in the art and may be produced using synthetic, semi -synthetic, or recombinant methods. Humanized acALY18 chimera antibodies may be advantageous for use in human subjects since substantially no secondary antibody reaction in human subjects may be caused.

[0073] In still other embodiments of the invention, fluorescently labeled acALY18, acALY18 peptides, or acALY18 antibodies may be made. In such embodiments, fluorescent dyes such as, but not limited to, phycoerythrin (PE), a red fluorescing dye, and fluorosceinisothiocynate (FITC), a green fluorescing dye, may be activated conjugated to the N- terminus of the peptidyl portion of acALY18, a free sulfhydryl or amino or carboxyl in the acALY18 peptide, or a free amino group of a acALY18 antibody.

[0074] Methods for making such conjugates are well known in the art for example, a acALY18 or acALY18 peptide may be conjugated to a fluorescent dye through its N-terminus by activating the peptide by attaching a thiol reactive extended -chain analogue of succinimidyltrans- 4-(maleimidylmethyl)cyclohexane-l-carboxylate(LC-SMCC) and separating unreacted LC- SMCC from the derivatized acALY18 peptide by size exclusion chromatography. The pyridyldisulfide derivative of PE or FITC to the free thiol may be activated by incubating the PE or FITC for 10 to 15 minutes in tris-(2-carboxyethyl)phosphine (TCEP). The purified LC- SMCC-acALY18 peptide derivative may then be combined with activated PE or FITC and mixed at 4° C overnight. The reaction may be stopped by the addition of N-ethylmaleimide (NEM) which caps any remaining thiol groups. The PE or FITC-acALY18 conjugate may be purified by size exclusion chromatography and lyophilized to yield the final product. [0075] In other embodiments of the invention, fluorescently labeled acALY18 or acALY18 peptides may be used to analyse tissue samples. For example, fluorescently labeled acALY18 or acALY18 peptides may be mixed ex vivo with samples of a subject to detect and quantitate cells engaging the fluorescently labeled acALY18 or acALY18 peptides. In still other embodiments, fluorescently labeled antibodies to the acALY18 or acALY18 peptides may be used to detect and quantitate the levels of acALY18 or acALY18 peptide in ex vivo samples from subjects using methods such as fluorescent microscopic methods, ELISA and the like. Such methods of analysis are well known to those practiced in the art.

[0076] The present invention can be further illustrated by reference to the following examples.

EXAMPLE 1

[0077] Primary human fibroblasts were cultured in 100 mm tissue culture plates at 37° C to 95% confluence. acALY18 was labeled with AlexaFluor 594 dye (Inviotrogen) following the manufacturer's recommended procedure and optimized to a 2:1 molar ratio (dye/peptide). Cells were incubated at room temperature with a 5 nM concentration of acALY 18 -AlexaFluor 594 for 15-20 minutes with gentle rocking and then examined using a fluorescence microsope (Nikkon Eclipse E2000U) at 40 X magnification.

[0078] Untreated cells were washed on ice with 2 X cold PBS and scrape harvested with 100 cold lysis buffer (0.5% TritonX/NET supplemented with 1 μΕ of Sigma

Protease Inhibitor). Cell lysate was spiked with 5 μg of acALY18 and analyzed by Western using a Tricine gel (Invitrogen) and transferred to a PVDF membrane for visualization using anti-acALY18 antibody. These results are presented in Figure 1.

[0079] Primary human fibroblasts were cultured with and without 25 nM

Thapsigargin for 1, 5, 15, 30, and 60 min, scrape harvested, lysed, analyzed by Western and probed for acALY18 using anti-acALY18 antibody. Significant induction of acALY18 was detected within 60 min (Figure 1 A). [0080] Primary human fibroblasts were cultured in the presence of 25 nM

Thapsigargin, 5 ng/mL acALY18, Chariot transfection reagent alone (mock), or untreated, scrape harvested, lysed, analyzed by Western and probed for the ER stress marker, GRP78. Only Thapsigargin treatment induced GRP78 after 24 h (Figure IB).

[0081] Primary human fibroblasts were cultured in the presence of 25 nM

Thapsigargin for 1, 5, 15, 30 60 min., and 24 h. Cells were scrape harvested , lysed, analyzed by Western, and probed for GRP78. No significant GRP78 expression was noted until the 24 h time point indicating acALY18 expression precedes GRP78 expression (Figure 1C).

EXAMPLE 2

[0082] TRPC ^" and wild type mouse embryonic fibroblasts were treated with 25 nM Thapsigargin for 1, 5, 15, 30, and 60 min. Cells were scrape harvested, lysed, analyzed by Western and probed for acALY18 and GAPDH (Figure 2 A and 2B). The results are normalized to GAPDH expression and show that TRPCl^{- -} fibroblasts do not show induction of acALY18 in response to Thapsigargin treatment (Figure 2C). IL-Ιβ expression was also measured in the wild type and TRPCl^{- -} fibroblasts in response to 72 h Thapsigargin treatment. No IL-Ιβ expression was induced in the TRPCl^{- -} fibroblasts (Figure 2D) thus establishing that acALY18 is derived from TRPC1.

EXAMPLE 3

[0083] Primary human fibroblasts were treated with 25 nM Thapsigargin (1, 5, 15, 30, and 60 min.), scrape harvested, lysed, analyzed by Western and probed for either acALY18 or XBP1 and GAPDH (Figure 3 A and 3B). The expression of acALY18 normalized to GAPDH (Figure 3C) shows > 3 -fold increase in acALY18 expression within 60 min and a 2-fold increase in XBP1 expression within 60 min (Figure 3D). acALY18 expression and XBPl expression appear at the same molecular mass (between 22 kDa and 36 kDa) suggesting that there may be an association between acALYl 8 and XBPl .

EXAMPLE 4

[0084] Primary human fibroblasts were treated with 25 nM Thapsigargin for 30 min., scrape harvested, lysed. The cell lysate was reduced with 50 mM DTT and 2.5% β- mercaptoethanol for 15, 30, or 60 min. and analyzed by Western and probed for acALY18 and GAPDH (Figure 4A). acALY18 expression was normalized to GAPDH and the acALY18 expression shows a linear decline with increasing exposure time to the DTT/BME reducing agent (60% after 60 min) as shown in Figure 4B. This result indicates that acALY18 association with its protein target (XBPl) is a covalent disulfide bond.

EXAMPLE 5

[0085] Primary human fibroblasts were incubated with 25 nM Thapsigargin for 60 min. or 24 h then harvested by scraping and concentrated into a cell pellet by centrifugation. Cells were lysed in TEN buffer (Tris/EDT A/NaC 1) with 0.5% Triton X.

[0086] Protein A coated magnetic beads were twice washed with PBS and incubated with XBPl antibody for 30 min. The beads were isolated by magnetic separation, washed 3 X with PBS, and stored in 100 PBS until the time of use.

[0087] 50 of whole cell lysate was pre-cleared by incubation for 20 min. with non-specific antibody (anti-IL-Ιβ). The pre-cleared lysate was collected after magnetic separation of the beads and transferred to a tube containing anti-XBPl antibody conjugated Protein A beads. The cell lysate/bead mixture was incubated at room temperature for 30 min. with gentle mixing. The beads were magnetically separated and washed 3 X with PBS. After the final wash the beads were suspended in 30 of SDS loading buffer and boiled for 30 minutes. The beads were magnetically separated and the supernatant was loaded onto Tris/gycine gels and the proteins were separated by electrophoresis at 140 voles for 120 min using a SDS/glycine running buffer. Proteins separated on the gel were transferred to a PVDF membrane using a transfer buffer (Invitrogen) supplemented with 10% methanol at 30 volts for 60 min.

[0088] The PVDF membrane was cut in half to probe for either acALY18 or XBP1. The membranes were blocked for 60 min. at room temperature with 5% skim milk powder dissolved in TBS.

[0089] XBP1 antibody was diluted 1 :500 in 3% bovine serum albumin in TBS and applied to one half of the membrane and acALY18 antibody was diluted 1 :1000 in 3% bovine serum albumin in TBS and applied to the other half of the membrane. Membranes were incubated overnight at 4° C with rocking. The membranes were then washed 3X in TBS for at least 20 min each wash. Both membranes were then incubated with donkey anti- rabbit horseradish peroxidase (1 :2000) in 3% bovine serum albumin/TBS for 3 h. The membranes were washed 3X in TBS for at least 20 min each wash and then incubated with 2 mL of ECL Chemiluminescence Reagent (Thermo Scientific) for 10 min. The immunoblots are depicted in Figure 5. A plot of the band intensity for the unspliced isoform of XBPl probed with acALY18 and probed with XBPl clearly shows an increasing expression of acALY18 expression associated with unspliced isoform of XBPl over the time course (Figure 5).

EXAMPLE 6

[0090] C57BL/6 murine fibroblasts were cultured to confluence in a T75 flask. Cells were incubated with 25 nM Thapsigargin for 60 min. then harvested by scraping and concentrated into a cell pellet by centrifugation. Cells were lysed in TEN buffer

(Tris/EDTA/NaCl) with 0.5% Triton X.

[0091] Two 50 portions of Protein A coated magnetic beads were twice washed with PBS. One aliquot was incubated with acALY18 antibody and the other with XBPl antibody for 30 min. The beads were isolated by magnetic separation, washed 3 X with PBS, and stored in 100 PBS until the time of use.

[0092] 50 μί of whole cell lysate was pre-cleared by incubation for 20 min. with 50 μΐ, of PBS washed Protein A beads not conjugated with an antibody. The pre-cleared lysate was collected after magnetic separation of the beads and transferred to a tube containing anti- acALY18 or anti-XBPl antibody conjugated Protein A beads. The cell lysate/bead mixture was incubated at room temperature for 30 min. with gentle mixing. The beads were magnetically separated and washed 3 X with PBS. After the final wash the beads were suspended in 30 μΐ, of SDS loading buffer and boiled for 30 minutes. The beads were magnetically separated and the supernatant was loaded onto a precast gel (Invitrogen) and the proteins were separated by electrophoresis at 140 voles for 120 min using a SDS/glycine running buffer. Proteins separated on the gel were transferred to a PVDF membrane using a transfer buffer (Invitrogen) supplemented with 10% methanol at 30 volts for 60 min.

[0093] The PVDF membrane was cut in half to separate the protein pulled out by ant- acALY18 antibody from that pulled out by anti-XBPl antibody. The membranes were blocked for 60 min. at room temperature with 5% skim milk powder dissolved in TBS.

[0094] XBP1 antibody was diluted 1 :500 in 3% bovine serum albumin in TBS and applied to the membrane containing proteins pulled out with anti-acALY18 antibody.

acALY18 antibody was diluted 1 :1000 in 3% bovine serum albumin in TBS and applied to the membrane containing proteins pulled out with anti-XBPl antibody. Membranes were incubated overnight at 4° C with rocking. The membranes were then washed 3X in TBS for at least 20 min each wash. Both membranes were then incubated with donkey anti -rabbit horseradish peroxidase (1 :2000) in 3% bovine serum albumin/TBS for 3 h. The membranes were washed 3X in TBS for at least 20 min each wash and then incubated with 2 mL of ECL Chemiluminescence Reagent (Thermo Scientific) for 10 min.

[0095] The results are shown in Figure 6 and clearly demonstrate that immobilized anti-acALY18 and anti-XBPl pulled out the same acALY18/XBPl complex. EXAMPLE 7

[0096] C57BL/6 murine fibroblasts were cultured to a cell density that was 95% confluent in three separate 60 mm tissue culture dishes. 0.5 of Plus Reagent (Invitrogen) was added to 50 μΕ of serum free Opti-MEM (Gibco) with either 4.89 μΕ acALY18 or 25 nM Thapsigargin and incubated for 5 min. at room temperature. Lipofectamine (1 μί) was added with mixing and the mixture incubated for 30 min. at room temperature. The lipofectamine solution without acALY18 or Thapsigargin was prepared as a control. The respective solutions (acALY18/lipofectamine, Thapsigargin/lipofectamine, or lipofectamine control) were added to separate culture dishes containing C57BL/6 fibroblasts and the cell cultures were incubate for 3 h at 37° C.

[0097] Cells from each culture were scrape -harvested into a 15-mL conical centrifuge tube and the cell culture dishes were twice washed with PBS and the washings were added to the respective tubes. A cell pellet was formed by centrifugation and the supernatants removed. Cells were lysed in 300 μΐ, RTL Buffer supplemented with β-mercaptoethanol. RNA was purified using the RNAeasy kit (Qiagen) according the manufacturer's instructions and RNA was converted to cDNA. 5.0 μί of the cDNA was subjected to PCR (35 cycles at 94°, 60°, and 72° C) separated on a 2% agarose gel and stained with ethidium bromide.

[0098] The results shown in Figure 8 clearly indicate that acALY18 and Thapsigargin reduce the expression of the unspliced isoform of XBP1 mRNA and increase the expression of the spliced isoform of XBP1 mRNA as compared to untreated controls.

EXAMPLE 8

[0099] Primary human fibroblasts were cultured in the presence of either 5 ng/mL acALY18 or 100 ng/mL LPS for 1, 4, or 24 h. Cells were scrape harvested, lysed, analyzed by Western and probed for XBP1 and GAPDH. Analysis of the expression of the unspliced isoform of XBPl normalized to GAPDH clearly shows the opposite effects of acALY18 and LPS (Figure 9). XBPlu expression declined nearly 3 -fold over the time course in the acALY18 treated cells and is consistent with the decline in XBPlu mR A result whereas LPS induced a 70% increase in XBPlu expression.

[0100] Although the present invention has been described with reference to specific embodiments, workers skilled in the art will recognize that many variations may be made therefrom, for example in the particular experimental conditions herein described, and it is to be understood and appreciated that the disclosures in accordance with the invention as set forth herein show only some preferred embodiments and objects and advantages of the invention without departing from the broader scope and spirit of the invention. It is to be understood and appreciated that these discoveries in accordance with this invention are only those which are illustrated of the many additional potential applications of the compounds and methods described, that may be envisioned by those of ordinary skill in the art, and thus are not in any way intended to be limiting of the invention. Accordingly, many other objects and advantages of the invention will be apparent to those skilled in the art from the detailed description herein together with the appended claims.

Claims

What Is Claimed Is:

1. A peptide or peptide fragment with the amino acid sequence

XLYDKGYTSKEQKDCVGIX or XLYDKGYTPKEQKDCVGIX or inversions or mimetics thereof, wherein X is absent or a naturally occurring amino acid or mimetic thereof, a derivatized amino acid or a non-amino acid prosthetic group, and that binds to one or both iso forms of X Box Protein 1 (XBP1 or XBPlu).

2. A peptide or peptide fragment with the amino acid sequence

XLYDKGYTSKEQKDCVGX or XLYDKGYTPKEQKDCVGX or inversions or mimetics thereof, wherein X is absent or a naturally occurring amino acid or mimetic thereof, a derivatized amino acid or a non-amino acid prosthetic group, and that binds to one or both iso forms of X Box Protein 1 (XBP1 or XBPlu).

3. The peptide or peptide fragment of claim 1, wherein the peptide or peptide fragment binds to XBP1 in a molar ratio of 1 : 1.

4. The peptide or peptide fragment of claim 1, wherein the peptide or peptide fragment binds to XBP1 in a molar ratio of 2: 1.

5. The peptide or peptide fragment of claim 1, wherein the peptide or peptide fragment binds to XBP1 in a molar ratio of 3 : 1.

6. The peptide or peptide fragment of claim 2, wherein the peptide or peptide fragment binds to XBP1 in a molar ratio of 1 : 1.

7. The peptide or peptide fragment of claim 2, wherein the peptide or peptide fragment binds to XBP1 in a molar ratio of 2: 1.

8. The peptide or peptide fragment of claim 2, wherein the peptide or peptide fragment binds to XBP1 in a molar ratio of 3 : 1.

9. The peptide or peptide fragment of claim 1, wherein the peptide or peptide fragment binds one or more of the cysteine amino acids at positions 204, 215 and 247 in XBPlu to form one or more disulfide bonds that covalently link the peptide or peptide fragment to XBPlu.

10. The peptide or peptide fragment of claim 2,wherein the peptide or peptide fragment binds one or more of the cysteine amino acids at positions 204, 215 and 247 in XBPlu to form one or more disulfide bonds that covalently link the peptide or peptide fragment to XBPlu.

11. The peptide or peptide fragment of claim 1 , wherein binding of the peptide or peptide fragment with XBPl induces assembly and activation of one or more of the intracellular inflammasomes .

12. The peptide or peptide fragment of claim 11, wherein the intracellular inflammasome is the NLRP3 inflammasome, thereby to induce an immune response and more specifically an innate immune response in an animal.

13. The peptide or peptide fragment of claim 2, wherein binding of the peptide or peptide fragment with XBPl induces assembly and activation of one or more of the intracellular inflammasomes .

14. The peptide or peptide fragment of claim 13, wherein the intracellular inflammasome is the NLRP3 inflammasome, thereby to induce an immune response and more specifically an innate immune response in an animal.

15. The peptide or peptide fragment of claim 12, wherein the immune response is induced for from about 1-3 days following administration of the the peptide or peptide fragment.

16. The peptide or peptide fragment of claim 14, wherein the immune response is induced for from about 1-3 days following administration of the peptide or peptide fragment.

17. The peptide or peptide fragment of claim 12, wherein the immune response is induced for about one week or longer following administration of the peptide or peptide fragment.

18. The peptide or peptide fragment of claim 14, wherein the immune response is induced for about one week or longer following administration of the peptide or peptide fragment.

19. A method for the prevention of onset or progression of a disease in a subject, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 1 to said subject prior to exposure to a disease causing agent or following exposure to a disease causing agent thereby to substantially prevent or arrest disease progression.

20. A method according to claim 19, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 1 to said subject following disease onset to lessen the severity of the disease or reverse the course of the disease.

21. A method for the prevention of onset or progression of a disease in a subject, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 2 to said subject prior to exposure to a disease causing agent or following exposure to a disease causing agent thereby to substantially prevent or arrest disease progression.

22. A method according to claim 21, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 2 to said subject following disease onset to lessen the severity of the disease or reverse the course of the disease.

23. A method for treating an infection in a subject, comprising administering a

physiologically effective amount of the peptide or peptide fragment according to claim 1 to said subject.

24. A method for treating an infection in a subject, comprising administering a

physiologically effective amount of the peptide or peptide fragment according to claim 2 to said subject.

25. A method for modulating an immune response in a subject, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 1 to said subject.

26. A method for modulating an immune response in a subject, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 2 to said subject.

27. A method for preventing a secondary infection in a subject, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 1 to said subject.

28. A method for preventing a secondary infection in a subject, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 2 to said subject.

29. A method for inhibiting inflammasome dependent expression of collagen in fibrotic diseases selected from the group consisting of systemic scleroderma, chronic obstructive pulmonary disease, and cystic fibrosis, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 1 to a subject exhibiting such inflammasome dependent expression.

30. A method for inhibiting inflammasome dependent expression of collagen in fibrotic diseases selected from the group consisting of systemic scleroderma, chronic obstructive pulmonary disease, and cystic fibrosis, comprising administering a physiologically effective amount of the peptide or peptide fragment according to claim 2 to said subject exhibiting such inflammasome dependent expression.

31. A method for treating inflammation in a subject exhibiting said inflammation selected from the group consisting of systemic inflammation, chronic inflammatory diseases, inflammation due to sepsis, non-septic injury, trauma, surgery and combinations thereof, which method comprises administering a physiologically effective amount of the peptide or peptide fragment according to claim 1 to said subject exhibiting such inflammation.

32. A method for treating inflammation in a subject exhibiting said inflammation selected from the group consisting of systemic inflammation, chronic inflammatory diseases, inflammation due to sepsis, non-septic injury, trauma, surgery and combinations thereof, which method comprises administering a physiologically effective amount of the peptide or peptide fragment according to claim 2 to said subject exhibiting such inflammation.

33. A method as claimed in any of claims 19 through 32, comprising administering the

physiologically effective amount of the peptides or peptide fragments to a subject by techniques selected from the group consisting of enteral, parenteral, and topical delivery.

34. A method as claimed in claim 33, wherein the physiologically effective amount of the peptides or peptide fragments are delivered directly to said subject.

35. A method as claimed in claim 33, wherein the physiologically effective amount of the peptides or peptide fragments are combined with a pharmaceutically-acceptable carrier to make a pharmaceutical composition that may be delivered or provided to said subject.