WO2018204976A1 - Anti-inflammatory agents and methods of treatment - Google Patents

Abstract

Disclosed are agents for treating or preventing inflammatory diseases and/or conditions. More particularly, the present invention discloses the use of ICAM-1 antagonists, including ICAM-1 antigen-binding molecules, for treating inflammatory diseases including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). In specific embodiments, the ICAM-1 antagonists are used in combination with immunotherapeutic agents to inhibit an inflammatory immune response.

Description

TITLE OF THE INVENTION

"ANTI-INFLAMMATORY AGENTS AND METHODS OF TREATMENT"

FIELD OF THE INVENTION

[0001] This application claims priority to Australian Provisional Application No.

2017901713 entitled "Anti-inflammatory agents and methods of treatment" filed 9 May 2017, the contents of which are incorporated herein by reference in their entirety.

[0002] This invention relates generally to agents for treating or preventing

inflammatory diseases and/or conditions. More particularly, the present invention relates to the use of ICAM-1 antagonists, including ICAM-1 antigen-binding molecules, for treating inflammatory diseases including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) . In specific embodiments, the ICAM-1 antagonists are used in combination with i mmunotherapeutic agents to inhibit an inflammatory immune response.

BACKGROUND OF THE INVENTION

[0003] The liver is the principal site of iron-related injury with progressive iron deposition leading to fibrosis, cirrhosis and hepatocellular carcinoma ( HCC) {see, Niederau et a/. , 1996) . Cirrhosis is the most important adverse prognostic factor with a five year survival if left untreated . Iron removal before the onset of cirrhosis restores normal life expectancy, and also results in reversal of fibrosis, but not cirrhosis {see, Niederau et a/., 1985; and Powell et a/., 2006) .

[0004] Tissue ferritin is an i mportant site for iron storage in a non-toxic biologically available form . It is comprised of 24 subunits of two subunit types: H-chain and L-chain ferritin (H- Ferritin and L-Ferritin, respectively) . H-Ferritin can regulate i mmune function {see, Olynyk et a/. , 1999; and Moss et a/. , 1992), hematopoiesis {see, Broxmeyer et a/. , 1989), and cell differentiation {see, Matzner eta/., 1979) . In haemochromatosis, serum ferritin is measured as an indicator of body iron stores and when grossly elevated is indicative of cirrhosis {see, Waalen et a/. , 2008) .

[0005] Chronic liver diseases leading to cirrhosis are commonly associated with inflammation and elevated ferritin and it has recently been determined that ferritin acts as a proinflammatory mediator in activated hepatic stellate cells (HSCs) or liver myofibroblasts that derive from activated HSCs , signaling via the PKC-(/N FKB pathway, and in doing so upregulating proinflammatory molecules associated with HSC activation and fibrogenesis {see, Ruddell et a/., 2009) . Tissue derived H-Ferritin activates an iron-independent signaling cascade involving PI-3 kinase, PKC-ζ, MAP-kinase/MEKK and ΙΚΚα/β, and resulting in upregulated N FKB expression, nuclear localization and increased DNA binding activity in HSC {see, Ruddell et a/. , 2009) .

[0006] Sterile inflammation {i.e., in the absence of pathogens) occurs during many chronic liver diseases in response to tissue stress and/or injury, and is a major factor in the development of both fibrosis and cancer. Upon tissue damage, damage-associated molecular patters (DAMPs) act on patter recognition receptors ("Signal 1") on nearby effector cells, such as immune/inflammatory cells (including HSCs), to induce the inflammasome, a cytosolic protein complex that activates caspase-1 (via "Signal 2") . This results in the secretion of IL-Ιβ, IL-18 and other cytokines. Notably, the effect of H-Ferritin on HSC biology causes a >200-fold increase in IL- 1β expression {see, Ruddell et a/., 2009) . [0007] Recently, several molecules have been proposed as potential H-Ferritin receptors, including T-cell immunoglobulin-domain and mucin-domain 2 (Tim-2) on B-cells {see, Chen eta/., 2005). Other studies have also identified Transferrin Receptor-1 (TfRl) on B-cells and T-cells, and H-Kininogen (HKa) as H-Ferritin binding partners {see, Li eta/., 2010; and

Parthasarathy eta/., 2002). However, the extent to which they play a role in inflammation is currently not known.

[0008] Although a role for HSCs in fibrosis associated with iron overload in animal models and in human haemochromatosis {see, Ramm eta/., 1995; and Ramm eta/., 1997) has been demonstrated, the precise pathophysiological mechanisms regulating fibrogenesis are not well understood. Thus, in order to develop effective treatments for inflammatory diseases {e.g., chronic liver disease), a better understanding of the mechanism of H-Ferritin-mediated inflammation is crucial.

SUMMARY OF THE INVENTION

[0009] The present invention is predicated in part on the determination that

Intercellular Adhesion Molecule 1 (ICAM-1) is a cell surface receptor for H-Ferritin, and the interaction between ICAM-1 and H-Ferritin activates a proinflammatory signaling cascade in HSCs. Furthermore, it was determined that such proinflammatory signaling relies on H-Ferritin internalization and endosome acidification . Based on this determination, the present inventors propose inhibiting or blocking H-Ferritin internalization using ICAM-1 antagonist to reduce aberrant proinflammatory signaling in HSCs and myofibroblasts or myofibroblast-like cells, including in the treatment of inflammatory diseases or conditions, as described hereafter.

[0010] Accordingly, in one aspect, the present invention provides methods of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide. These methods generally comprise contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell.

[0011] In another aspect, the present invention provides methods of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide. Methods relating to this aspect comprise contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, thereby inhibiting inflammasome assembly in the cell . Typically, the inflammasome comprises at least one {e.g., 1, 2, 3, etc.) component selected from caspase-1 (Casp-1), apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain (ASC), and NOD-like receptors. In some embodiments, the inflammasome comprises at least one {e.g., 1, 2, 3, 4, 5, etc.) of Casp-1, ASC, NOD-like receptor containing pyrin domain 3 (NLRP3), NOD-like receptor containing pyrin domain 1 (NLRP1) and NLR family CARD domain-containing protein 4 (NLRC4). The cell that comprises a cell surface ICAM-1 polypeptide is suitably an hepatic stellate cell (HSC) or a myofibroblast. Suitably, the NOD-like receptors are selected from NLRP3, or NLRP1 and NLRC4.

[0012] In some embodiments, the ICAM-1 antagonist binds to a specific region of the ICAM-1 polypeptide, representative examples of which include domain 1 {e.g., residues 1 to 83; or residues 1 to 84, as disclosed by Bella eta/., 1998; also referred to herein as Dl), domain 2 {e.g., residues 84 to 185; or residues 85 to 185, as disclosed by Bella eta/., 1998; also referred to herein as D2), domain 3 {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998; also referred to herein as D3), domain 4 {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998; also referred to herein as D4) of the ICAM-1 polypeptide, and domain 5 {e.g., residues 367 to 450; or residues 386 to 453, as disclosed by Bella eta/., 1998; also referred to herein as D5) of the ICAM-1 polypeptide, wherein the numbering of the residues is relative to the amino acid numbering of the mature polypeptide, as set forth for example in SEQ ID NO: 1.

[0013] Accordingly, in related aspects, the present invention provides methods of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 1 {e.g., residues 1 to 83; or residues 1 to 84, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell.

[0014] In other related aspects, the present invention provides methods of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 2 {e.g., residues 84 to 185; or residues 85 to 185, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell .

[0015] In still other related aspects, the present invention provides methods of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM- 1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 3 {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell .

[0016] Yet other related aspects of the present invention provide methods of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 4 {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell .

[0017] Still other related aspects of the present invention provide methods of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 5 {e.g., residues 367 to 450; or residues 386 to 453, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell .

[0018] In further related aspects, the present invention provides methods of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 1 of ICAM-1 {e.g., residues 1 to 83; or residues 1 to 84, as disclosed by Bella eta/., 1998), thereby inhibiting inflammasome assembly in the cell, and/or inhibiting inflammasome assembly in the cell .

[0019] In other related aspects, the present invention provides methods of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 2 of ICAM-1 {e.g., residues 84 to 185; or residues 85 to 185, as disclosed by Bella eta/., 1998), thereby inhibiting inflammasome assembly in the cell, and/or inhibiting inflammasome assembly in the cell .

[0020] In still other related aspects, the present invention provides methods of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 3 of ICAM-1 {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998), thereby inhibiting inflammasome assembly in the cell, and/or inhibiting inflammasome assembly in the cell.

[0021] In still other related aspects, the present invention provides methods of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 4 of ICAM-1 {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998), thereby inhibiting inflammasome assembly in the cell, and/or inhibiting inflammasome assembly in the cell.

[0022] In further related aspects, the present invention provides methods of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 5 of ICAM-1 {e.g., residues 367 to 450; or residues 386 to 453, as disclosed by Bella eta/., 1998), thereby inhibiting inflammasome assembly in the cell, and/or inhibiting inflammasome assembly in the cell.

[0023] Suitably, in any of the aspects broadly described above and elsewhere herein, the ICAM-1 antagonist does not substantially inhibit the interaction between the ICAM-1 polypeptide and a macrophage 1 antigen (MAC-1) polypeptide. Alternatively, or in addition, the ICAM-1 antagonist does not substantially inhibit the interaction between an ICAM-1 polypeptide and a lymphocyte function-associated antigen 1 (LFA-1) polypeptide. Alternatively, or in addition, the ICAM-1 antagonist does not substantially inhibit the interaction between an ICAM-1 polypeptide and a vascular cell adhesion protein 1 (VCAM-1) polypeptide.

[0024] Suitably, in any of the aspects broadly described above and elsewhere herein, the methods reduce the expression in the cell of at least one inflammatory cytokine {e.g., an IL-1 family cytokine) . In non-limiting examples of this type, the at least one inflammatory cytokine comprises interleukin-ΐβ (IL-Ιβ).

[0025] Suitably, in any of the aspects broadly described above and elsewhere herein, the ICAM-1 antagonist is an antigen-binding molecule that binds to a specific region of an ICAM-1 polypeptide. Suitable antigen-binding molecules for use in the methods described herein include, but are not limited to, a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody. In some preferred embodiments the antigen-binding molecule is a monoclonal antibody.

[0026] In some embodiments, the antigen-binding molecule binds to a specific region of the ICAM-1 polypeptide that is located fully or at least partially within domain 1 {e.g., residues 1 to 83; or residues 1 to 84, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide. In some embodiments, the antigen-binding molecule binds to a specific region of the ICAM-1 polypeptide that is located fully or at least partially within domain 2 {e.g., residues 84 to 185; or residues 85 to 185, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide. In some embodiments, the antigen-binding molecule binds to a specific region of the ICAM-1 polypeptide that is located fully or at least partially within domain 3 {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide. In some embodiments the antigen- binding molecule binds to a specific region of the ICAM-1 polypeptide that is located fully or at least partially within domain 4 {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide. In some embodiments, the antigen-binding molecule binds to a specific region of the ICAM-1 polypeptide that is located fully or at least partially within domain 5 {e.g., residues 367 to 450; or residues 386 to 453, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide. In some embodiments, the antigen-binding molecule binds to a specific region of the ICAM-1 polypeptide that is located at least partially within domain 3 {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998) and at least partially within domain 4 {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide.

[0027] Still in another aspect of the present invention provides methods of producing an anti-inflammatory agent. These production methods generally comprise: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an antiinflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

Suitably, the methods further comprise a step of derivatizing the anti-inflammatory agent, and optionally, formulating the derivatized anti-inflammatory agent with a pharmaceutically acceptable carrier and/or diluent, to improve the efficacy of the agent or derivatized agent for inhibiting the inflammatory response. The cell that comprises a cell surface ICAM-1 polypeptide is preferably a HSC, or a myofibroblast or myofibroblast-like cell .

[0028] In some embodiments of this aspect, the detection of an inflammatory response includes the step of measuring the production of at least one inflammatory cytokine by the cell . By way of an example, the at least one inflammatory cytokine is IL-Ιβ.

[0029] In some of the same and other embodiments, the detection of an inflammatory response includes the step of measuring internalization of the H-Ferritin polypeptide into the cell . In representative methods of this type, the inflammatory response is detected as being inhibited if internalization of the H-Ferritin polypeptide is reduced.

[0030] In related aspect, the present invention provides methods of producing an antiinflammatory agent comprising : (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 1 (residues 1 to 83) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0031] In other related aspects, the present invention provides methods of producing an anti-inflammatory agent comprising: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 2 (residues 84 to 185) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0032] Still other related aspects of the present invention provide methods of producing an anti-inflammatory agent comprising: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 3 (residues 186 to 281) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0033] In yet other related aspects, the present invention provides methods of producing an anti-inflammatory agent comprising: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 4 (residues 282 to 366) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0034] In still In other related aspects, the present invention provides methods of producing an anti-inflammatory agent comprising: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 5 (residues 367 to 450) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0035] In each of the production methods broadly described above and elsewhere herein, the methods may further comprise derivatizing the anti-inflammatory agent, and optionally formulating the derivatized anti-inflammatory agent with a pharmaceutically acceptable carrier and/or diluent, to improve the efficacy of the agent or derivatized agent for inhibiting the inflammatory response.

[0036] Any suitable method of detecting an inflammatory response can be used in the methods disclosed above and elsewhere herein . For example, in some embodiments the antiinflammatory response is detected by a method comprising measuring the production of one or more inflammatory cytokines by the cell . In some embodiments the one or more inflammatory cytokines may comprise IL-Ιβ. In some of the same and other embodiments, the detection of an inflammatory response includes the step of measuring internalization of the H-Ferritin polypeptide into the cell . In illustrative examples of this type, the inflammatory response is detected as being inhibited if the internalization of the H-Ferritin polypeptide is reduced.

[0037] In preferred embodiments the cell is an HSC, or a myofibroblast or

myofibroblast-like cell .

[0038] In some of the same and other embodiments the candidate ICAM-1 antagonist is an antigen-binding molecule (for example, a monoclonal antibody, single-chain Fv (scFv), Fab fragment, F(ab') fragment, intrabody, and synthetic antibody). In some preferred embodiments, the antigen-binding molecule is a monoclonal antibody.

[0039] In some embodiments, the antigen-binding molecule binds to a different site on the ICAM-1 polypeptide, and therefore does not compete with the R6.5 antibody (ATCC deposit no. HB-9580) for binding to the ICAM-1 polypeptide.

[0040] In still yet another aspect, the present invention provides methods of screening for an antagonist of binding between an ICAM-1 polypeptide and an H-Ferritin polypeptide. The methods of this aspect generally comprise incubating an ICAM-1 polypeptide and an H-Ferritin polypeptide in the presence of a candidate agent, and detecting whether the candidate agent inhibits binding of the ICAM-1 polypeptide with the H-Ferritin polypeptide, which indicates that the candidate agent is an antagonist of the binding between an ICAM-1 polypeptide and an H-Ferritin polypeptide. In some embodiments, the ICAM-1 polypeptide is located on the surface of a cell {e.g., an HSC or a myofibroblast or myofibroblast-like cell).

[0041] The detecting step in these methods typically comprises measuring inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of a candidate agent. In some embodiments, the detection step comprises quantifying the production of one or more inflammatory cytokines (for example, IL-Ιβ and/or IL-18).

[0042] In another aspect, the present invention provides methods of producing an antigen-binding molecule {e.g., a monoclonal antibody) that binds specifically to at least one region of an ICAM-1 polypeptide, as broadly defined above and elsewhere herein {e.g., to one or more of domains 1 to 5 of an ICAM-1 polypeptide) and that inhibits at least one inflammatory activity selected from : (i) an inflammatory response produced by a cell that comprises a cell surface ICAM- 1 polypeptide, or (ii) internalization of H-ferritin into a cell that comprises a cell surface ICAM-1 polypeptide, and (iii) inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, comprising: (1) generating an antigen-binding molecule that binds to an ICAM-1 polypeptide {e.g., by immunizing an animal with an immunizing polypeptide comprising an amino acid sequence corresponding to an ICAM-1 polypeptide or to at least one region of an ICAM-1 polypeptide as broadly described above and elsewhere herein^.^., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 contiguous amino acids of an ICAM -1 polypeptide domain); identifying and/or isolating a B cell from the animal, which is immuno- interactive with the immunizing polypeptide or at least one region thereof; and producing the antigen-binding molecule expressed by that B cell); (2) contacting a cell that comprises a cell surface ICAM-1 polypeptide with the antigen-binding molecule, in the presence of an H-Ferritin polypeptide; and (3) detecting inhibition of an inflammatory response produced by the cell, or internalization of H-ferritin into the cell, or inflammasome assembly in the cell, as compared to the inflammatory response, internalization of H-ferritin, or inflammasome assembly in the absence of the antigen-binding molecule, which indicates that the antigen-binding molecule inhibits said at least one of inflammatory activity. In some embodiments, the methods comprise synthesizing or otherwise producing the antigen-binding molecule.

[0043] Also provided as a further aspect of the invention is an antigen-binding molecule produced by the generation methods broadly described above and elsewhere herein, or a derivative antigen-binding molecule with the same epitope-binding specificity as the antigen- binding molecule. The derivative antigen-binding molecule may be selected from antibody fragments (such as Fab, Fab', F(ab')₂, Fv), single chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen

binding/recognition site.

[0044] In still another aspect, the present invention provides methods of treating an inflammatory disease or condition in a subject. These methods generally comprise administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, thereby treating the inflammatory disease or condition. In some preferred embodiments, the ICAM-1 antagonist is an ICAM-1 antigen-binding molecule {e.g., a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody).

[0045] In some embodiments, the ICAM-1 antagonist {e.g., antigen-binding molecule) binds to a region located fully or at least partially within domain 1 {e.g., residues 1 to 83; or residues 1 to 84, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide. In some of the same embodiments and other embodiments, the ICAM-1 antagonist {e.g., antigen-binding molecule) binds to a region located fully or at least partially within domain 2 {e.g., residues 84 to 185; or residues 85 to 185, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide. In some of the same embodiments and other embodiments, the ICAM-1 antagonist {e.g., antigen-binding molecule) binds to a region located fully or at least partially within domain 3 {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide. In some embodiments, the ICAM-1 antagonist {e.g., antigen-binding molecule) binds to a region located fully or at least partially within domain 4 {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide. In some embodiments, the ICAM-1 antagonist {e.g., antigen-binding molecule) binds to a region located fully or at least partially within domain 5 {e.g., residues 367 to 450; or residues 386 to 453, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide.

[0046] In some embodiments, the ICAM-1 antagonist {e.g., antigen-binding molecule) binds to a different region to, and therefore does not compete for binding to the ICAM-1 polypeptide with the R6.5 antibody (ATCC deposit no. HB-9580).

[0047] In specific embodiments, the inflammatory disease or condition is a chronic liver disease, illustrative examples of which include fibrosis and/or cirrhosis. In other embodiments, the inflammatory disease or condition is a carcinoma {e.g., hepatocellular carcinoma (HCC)).

[0048] Sill in another aspect the present invention provides methods of treating an inflammatory disease or condition in a subject. These methods generally comprise administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 1 {e.g., residues 1 to 83; or residues 1 to 84, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby treating the inflammatory disease or condition in the subject.

[0049] In yet another aspect, the present invention provides methods of treating an inflammatory disease or condition in a subject. The methods of this aspect generally comprise administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 2 {e.g., residues 84 to 185; or residues 85 to 185, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby treating the inflammatory disease or condition in the subject.

[0050] Still yet another aspect of present invention provides methods of treating an inflammatory disease or condition in a subject. These methods generally comprise administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 3 {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby treating the inflammatory disease or condition in the subject.

[0051] In another aspect, the present invention provides methods of treating an inflammatory disease or condition in a subject. These methods generally comprise administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 4 {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby treating the inflammatory disease or condition in the subject.

[0052] Yet another aspect of the present invention provides methods of treating an inflammatory disease or condition in a subject. Methods in accordance with this aspect generally comprise administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 5 {e.g., residues 367 to 450; or residues 386 to 453, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby treating the inflammatory disease or condition in the subject.

[0053] In some embodiments of this type, the ICAM-1 antagonist is an antigen-binding molecule. For example, antigen-binding molecules that are particularly suitable for use with the present invention may be selected from a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody.

[0054] In some embodiments, the inflammatory disease or condition is a chronic liver disease, illustrative examples of which include fibrosis and/or cirrhosis. In other embodiments, the inflammatory disease or condition is a carcinoma {e.g., HCC).

[0055] In still another aspect the present invention provides the use of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, or an anti-inflammatory agent produced by the production methods broadly described above and elsewhere herein, in the manufacture of a medicament for the treatment of chronic liver disease (for example, fibrosis and cirrhosis) or a carcinoma {e.g., HCC).

[0056] In yet another aspect the present invention provides antigen-binding molecules, that are suitably purified or isolated, for use in reducing inflammation in a subject. Generally, the antigen-binding molecules binds to an ICAM-1 polypeptide and inhibit the binding of the ICAM-1 polypeptide to an H-Ferritin polypeptide. In some embodiments of this type the antigen-binding molecules do not substantially inhibit the binding of the ICAM-1 polypeptide to one or both of a

LFA1 polypeptide and a MAC-1 polypeptide. Such antigen-binding molecules may be selected from a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody. In some embodiments, the antigen-binding molecules are suitably human, humanized or chimeric antibodies.

[0057] In some embodiments, the antigen-binding molecules do not substantially bind to an ICAM-2 polypeptide. In some of the same embodiments and other embodiments, the antigen-binding molecule does not substantially bind to an ICAM-3 polypeptide. In some of the same embodiments and other embodiments, the antigen-binding molecule does not substantially bind to an ICAM-5 polypeptide. In some of the same embodiments and other embodiments, the antigen-binding molecule does not substantially bind to a VCAM-1 polypeptide.

[0058] In another aspect the present invention provides an inflammatory agent produced by the production methods broadly described above and elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is a photographic and graphical representation showing that H-Ferritin stimulates inflammasome activation in HSC. a. Stimulation of HSC with FTHl (4hours, 10 nM) up- regulates NLRP3 protein levels and Caspase-1 cleavage (represented by Caspase-1 peptide 20kDa, p20). b. FTHl (4hours, 10 nM) up-regulates NLRP3 protein levels but not NLRP1. In Figl.a and Figl.b actin was used as a loading control. We used 20 pg of protein/condition, c. FTHl (24hours, 10 nM) increases the levels of ASC at similar levels than LPS (24hours, 100 ng/mL). d. In response to FTHl (24hours, 10 nM) and LPS (24hours, 100 ng/mL) there is an augment in the colocalization between ASC (red) and ILip (green) in HSC. e. NLRP3 and ASC western-blot in siRNA-induced NLRP1, NLRP3 or NLRC4-deficient HSC. f. ELISA analysis of the ILip secretion (showed as fold change) in siRNA-induced NLRP1, NLRP3 or NLRC4-deficient HSC. g. ILip expression in Huh7 hepatoma cells treated (2hours, lOnM) with recombinant FTHl and FTLl. h. ΙΙ_1β gene expression in ex vivo" FTHl (12hours, 10 nM) and LPS (12hours, 100 ng/mL)-treated mouse liver slides. Groups were compared using one-way analysis of variance (ANOVA) with Dunnett's multiple comparison post hoc test or the Student ."test, where applicable using Prism 4.0 (GraphPad Software). Results are presented as mean (fold change) ± SEM. P < 0.05. *; P < 0.01. **; P < 0.005. ***; P < 0.001; **** was considered statistically significant.

[0060] Figure 2 is a photographic and graphical representation showing that H-Ferritin stimulates inflammasome activation in HSC. a. SDS-electrophoresis gel showing bands containing Alexa488-labeled FTHl. In the right side are described the proteins identified by mass

spectrometry in the Alexa488-labeled FTHl gel bands, b. Proteins identify by Ligand-receptor

Glycocapture that bind to FTHl and emerge as potential candidates for FTHl-receptor in rat HSC. Enlarge square of the Ligand-receptor Glycocapture outcomes highlights ICAM-las the only statistical significant FTHl-binding protein, c. ILip gene expression in non-treated and FTH1- treated HSCs (2hours, 10 nM) overexpressing GFP or ICAM l-GFP. d. ILip gene expression in scramble (SCR) and siRNA ICAM-l-transfected HSCs in response to vehicle (PBS) and FTHl

(2hours, lOnM). e. NLRP3 western-blot in scramble (SCR) and siRNA ICAM-l-trasfected HSCs in response to vehicle (PBS) and FTHl (24hours, lOnM). Actin was used as a loading control . We used 20ug of protein/condition, f. ELISA analysis of the ILip secretion (showed as fold change) in scramble (SCR) and siRNA ICAM-l-transfected HSCs in response to vehicle (PBS) and FTHl (24hours, lOnM). g. ILip gene expression in non-treated vs. FTHl-treated (2hours, lOnM) and FTHl + GFP-ICAM-l-overexpressing HSCs, treated with the clathrin-coated pit inhibitor Pitstop™ (Pit). Groups were compared using one-way analysis of variance (ANOVA) with Dunnett's multiple comparison post hoc test or the Student ."test, where applicable using Prism 4.0 (GraphPad Software). Results are presented as mean (fold change) ± SEM. P < 0.05. *; P < 0.01. **; P < 0.005. ***; P < 0.001; **** was considered statistically significant.

[0061] Figure 3 is a photographic representation showing that FTHl stimulates inflammasome activation in HSCs. a. Western-blot showing that FTHl (10 nM, 4 hours) up- regulates NLRP3 protein levels but not NLRP1, and induces both pro- and active (cleaved) ILip protein.

[0062] Figure 4 is a photographic representation depicting the role of ICAM-1 in FTH-l- induced inflammasome activation. Western-blot in scrambled (SCR) and siRNA ICAM-l-transfected HSCs in response to vehicle (PBS, Ctrl) and FTHl (10 nM, 24 hours). Data shows complete inhibition of FTH-l-induced NLRP3 + pro-Caspase-1 protein expression, as well as marked inhibition of active (cleaved) Caspase-1 protein expression in HSC lacking ICAM-1 (n=3).

[0063] Figure 5 is a schematic and graphical representation showing the role of ICAM-1 domains in FTH-l-induced IL-Ιβ signaling. (A) ICAM-1 domains showing mutants generated based on exome domain sequences. (B) Expression of IL-lb mRNA in FTHl (10 nM, 4 hours) treated HSCs, following transfection of sequential ICAM-1 exome domain mutants. (n=4). (C) ICAM-1 domains showing mutants generated based on protein domain sequences. (D) Expression of IL-lb mRNA in FTHl (10 nM, 4 hours) treated HSCs, following transfection of sequential ICAM-1 protein domain mutants. (n=4). Data are expressed as fold change (± SEM) relative to full length ICAM-1 with vector control background expression subtracted to correct for endogenous ICAM-1 signaling. Statistics using ANOVA with Dunnett's test where *, p>0.05; ****, p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0064] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0065] The articles "a" and "an" are used herein to refer to one or to more than one {i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0066] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about." In this manner, slight variations above and below the stated ranges {e.g., less than or equal to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%) can be used to achieve substantially the same results as values within the ranges. Also, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. In embodiments in which the stated range defines the position of an amino acid residue, for example, at the beginning or end of a domain, the present invention encompasses the defined position as well as slight variations {e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids) upstream or downstream of that position.

[0067] The terms "administration concurrently" or "administering concurrently" or "coadministering" and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition. By "simultaneously" is meant that the active agents are administered at substantially the same time, and desirably together in the same formulation. By "contemporaneously" it is meant that the active agents are administered closely in time, e.g., one agent is administered within from one minute to within one day before or after another. Any contemporaneous time is useful . However, it will often be the case that when not administered simultaneously, the agents will be administered within one minute to within eight hours and suitably within less than one to four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term "same site" includes the exact location, but can be within 0.5 to 15 centimeters, preferably from within 0.5 to 5 centimeters . The term "separately" as used herein means that the agents are administered at an interval, for example at an interval of a day to several weeks or months. The active agents may be

administered in either order. The term "sequentially" as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.

[0068] The term "agent" refers to any compound or substance, or mixture of compounds or substances, which induces a desired pharmacological and/or physiological effect. The term also encompass pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the above term is used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "agent" is not to be construed narrowly but extends to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins as well as compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof as well as cellular agents. The term "agent" includes a cell that is capable of producing and secreting a polypeptide referred to herein as well as a polynucleotide comprising a nucleotide sequence that encodes that polypeptide. Thus, the term "agent" extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells.

[0069] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0070] By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen {e.g., an ICAM-1 polypeptide). It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. "Antigen-binding molecules" typically comprise at least a portion of an intact antibody comprising the antigen binding region thereof. Examples of antigen- binding molecules include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies; single- chain antibody molecules; and multispecific antibodies formed from antibody fragment(s).

[0071] The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies {e.g., bispecific antibodies), antibody fragments, or any other antigen-binding molecule so long as they exhibit the desired biological activity.

[0072] An antibody "that binds" an antigen of interest {e.g., an ICAM-1 polypeptide) is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a "non-target" protein will be less than about 10% of the binding of the antibody, oligopeptide or other organic molecule to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.

[0073] As used herein, the term "binds specifically" and the like when referring to an antigen-binding molecule refers to a binding reaction which is determinative of the presence of an antigen in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen-binding molecules bind to a particular antigen and do not bind in a significant amount to other proteins or antigens present in the sample. Specific binding to an antigen under such conditions may require an antigen-binding molecule that is selected for its specificity for a particular antigen. For example, antigen-binding molecules can be raised to a selected protein antigen, which bind to that antigen but not to other proteins present in a sample. A variety of immunoassay formats may be used to select antigen-binding molecules specifically immuno-interactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immuno-interactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0074] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0075] By "corresponds to" or "corresponding to" is meant an amino acid sequence that displays substantial sequence similarity or identity to a reference amino acid sequence {e.g., at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83; 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarity or identity to all or a portion of the reference amino acid sequence).

[0076] As used herein, the term "does not substantially bind" to a particular target, as used herein, means does not bind or does not bind with a high affinity to the target {e.g., molecule or cell), binds to the target with a K_D of 10^"5 M or more, preferably 10^"5 M or more, more preferably 10^"4 M or more, more preferably 10^"3 M or more, or even more preferably 10^"2 M or more.

[0077] As used herein, the term "does not substantially inhibit an interaction" is intended to mean that the ability of an ICAM-1 antagonist of the invention to inhibit the interaction between ICAM-1 and another molecule {e.g., MAC-1, LFA-1) is essentially no greater than about 20% inhibition, more preferably no greater than about 15% inhibition, more preferably no greater than about 10% inhibition, more preferably no greater than about inhibition 5%, more preferably no greater than about inhibition 1%, more preferably no greater than about 0.1 inhibition. In certain embodiments, an ICAM-1 antagonist that does not substantially inhibit an interaction is intended to mean that the ICAM-1 antagonist does not inhibit the interaction between a soluble ICAM-1 protein and another molecule {e.g., MAC-1, LFA-1) at a concentration at which an agent {e.g., a known antibody that inhibits the binding of ICAM-1 to the other molecule) is capable of inhibiting such an interaction. In certain embodiments, an ICAM-1 antagonist that does not substantially inhibit an interaction is intended to mean the ability of an ICAM-1 antagonist of the invention to inhibit the interaction between ICAM-1 and another molecule {e.g., MAC-1, LFA-1) is essentially no greater than the ability of an unrelated, control agent {e.g., an antigen-binding molecule that does not have measurable affinity for ICAM-1) to inhibit the interaction between ICAM-1 and the other molecule {e.g., MAC-1, LFA-1) . [0078] By "effective amount," in the context of treating or preventing a disease or condition {e.g., a chronic liver disease) is meant the administration of an amount of active agent to a subject, either in a single dose or as part of a series or slow release system, which is effective for the treatment or prevention of that disease or condition. The effective amount will vary depending upon the health and physical condition of the subject and the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.

[0079] As used herein, the term "function" refers to a biological, enzymatic, or therapeutic function.

[0080] The term "ICAM", "ICAM-1 polypeptide" and the like as used herein means

"Intercellular Adhesion Molecule l" a polypeptide having a sequence according to UniProt accession no. P05362, the product of the ICAM1 gene {e.g., the human ICAM1 gene identified by GenBank accession no. NC_018930, or NM_000201), and includes all of the variants, isoforms and species homologs of ICAM-1.

[0081] As used herein, the term "ICAM-1 antagonist" refers to any agent that inhibits or abrogates the interaction between ICAM-1 and H-Ferritin. For example, an ICAM-1 antagonist may compete with an agonist {e.g., H-Ferritin) or partial agonist for binding to ICAM-1, thereby inhibiting the action of the agonist or partial agonist on ICAM-1, including H-Ferritin internalization and/or inflammasome assembly. Thus, the term "ICAM-1 antagonist" refers to an agent that is capable of substantially reducing, inhibiting, blocking, and/or mitigating H-Ferritin internalization and/or the activation of H-Ferritin-ICAM-1 mediated inflammasome assembly and consequential production of pro-inflammatory mediators in a cell that comprises a surface ICAM-1 polypeptide. Inhibition of Ferritin internalization and/or the activation of H-Ferritin-ICAM-1 mediated

inflammasome assembly by an ICAM-1 antagonist of the present invention suitably reduces or inhibits the production of pro-inflammatory mediators including pro-inflammatory cytokines by the cell.

[0082] The term "interaction", when referring to an interaction between two molecules, refers to the physical contact of the molecules with one another. Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules. The physical contact typically requires binding or association of the molecules with one another and may involve the formation of an induced magnetic field or paramagnetic field, covalent bond formation, ionic interaction (such as, for example, as occurs in an ionic lattice), a hydrogen bond, or alternatively, a van der Waals interaction such as, for example, a dipole-dipole interaction, dipole-induced dipole interaction, induced dipole-induced dipole interaction, or a repulsive interaction, or any combination of the above forces of attraction.

[0083] The terms "isolated" and "purified", as used herein, refer to a material that is substantially or essentially removed from or concentrated in its natural environment and substantially free of contaminants that interfere with the function or activity of the material. For example, an isolated antigen-binding material is separated from other antigen-binding materials that bind to other antigens, and free from other biological materials {e.g., other nucleic acids, proteins, lipids, cellular components) with which it is naturally associated.

[0084] The term "reduced H-Ferritin polypeptide internalization" or "reduced internalization of H-Ferritin" refers to reduced or abrogated internalization of an H-Ferritin polypeptide into a cell that comprises an ICAM-1 polypeptide on the cell surface, as compared with internalization of the H-Ferritin polypeptide in a normal cell comprising a functional ICAM-1 polypeptide on the cell surface. In specific embodiments, an impaired or abrogated internalization of H-Ferratin polypeptide is indicated when, suitably after at least 10 minutes {e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more minutes) in the presence of an ICAM-1 polypeptide, at least 90% {e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the H-Ferritin polypeptide in a sample remains extracellular to the cells that comprise an ICAM-1 polypeptide, or localized to the plasma membrane {e.g., basolateral membrane localization) of such cells. By contrast, an "unimpaired H- Ferritin polypeptide internalization" or "unimpaired internalization of H-Ferritin polypeptide" refers to the same, similar or greater internalization of the H-Ferritin polypeptide into a cell that comprises an ICAM-1 polypeptide on the cell surface when the H-Ferritin is bound to the ICAM-1 polypeptide, as compared with internalization of the H-Ferritin polypeptide in a normal ICAM-1- expressing cell when the H-Ferritin polypeptide is bound to functional ICAM-1. In some

embodiments, an unimpaired ICAM-l-induced internalization of an H-Ferritin polypeptide is indicated when, suitably after at least 10 minutes {e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more minutes) in the presence of an ICAM-1 polypeptide, less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or even less of the H-Ferritin polypeptide in a sample remains extracellular to the cells that comprise an ICAM-1 polypeptide or are localized to the plasma membrane {e.g., basolateral membrane localization) of such cells.

[0085] As used herein, the terms "label" and "detectable label" refer to a molecule capable of being detected, where such molecules include, but are not limited to, radioactive isotopes, fluorescers (fluorophores), chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands {e.g., biotin, avidin, streptavidin or haptens), intercalating dyes and the like. The term "fluorescer" or

"fluorophore" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable range.

[0086] The term "monoclonal antibody" as used herein refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope(s), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds to a target {e.g., a target antigen), wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method {e.g., Kohler eta/., Nature, 256:495 (1975); Harlow eta/., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling eta/., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, N .Y., 1981)), recombinant DNA methods {see, e.g., U.S. Pat. No. 4,816,567), phage display technologies {see, e.g., Clackson eta/. (1991) Nature 352 : 624-628; Marks eta/. (1991) J. Mol.

Biol. 222: 581-597; Sidhu eta/. (2004) J. Mo/. Biol. 338(2) : 299-310; Lee eta/. (2004) J. Mo/. Biol. 340(5) : 1073-1093; Fellouse (2004) Proc. Nat. Acad. Sci. USA 101(34) : 12467-12472; and Lee et al. (2004) J. Immunol. Methods 284(1-2) : 119-132, and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences {see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90 : 2551;

Jakobovits et al. (1993) Nature 362 : 255-258; Bruggemann et al. (1993) Year in Immuno. 7: 33; U.S. Pat. Nos. 5,545,806; 5,569,825; 5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; WO 1997/17852; U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al. (1992) Bio/Technology 10 : 779-783; Lonberg et al. (1994) Nature l 856-859; Morrison (1994) Nature, 368: 812-813; Fishwild et al. (1996) Nature Biotechnology 14: 845-851; Neuberger (1996) Nature Biotechnology 14: 826; and Lonberg and Huszar (1995) Intern. Rev. Immunol. 13 : 65-93).

[0087] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to

corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81 : 6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate {e.g., Old World Monkey, Ape etc.) and human constant region sequences, as well as "humanized" antibodies.

[0088] "Humanized" forms of non-human {e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones eta/. (1986) Nature 321 : 522-525; Riechmann eta/. (1988) Nature 332 : 323- 329; and Presta (1992) Curr. Op. Struct. Biol. 2 : 593-596.

[0089] The term "myofibroblast" refers to fibroblasts that transitioned from fibroblast into fibroblasts that are differentiated towards a smooth muscle cell-like phenotype, and which express high levels of alpha smooth muscle actin (a-SMA) and are positive for a-SMA.

Myofibroblast are associated with the increased and often pathological deposition of extracellular matrix (ECM) at fibrotic lesions. Myofibroblasts are activated in response to injury or increased epithelial to mesenchymal crosstalk and are thought to be the primary producers of ECM components following injury. Myofibroblasts originate from differentiation of resident mesenchymal fibroblasts (hepatic stellate cells in the liver), from epithelial to mesenchymal transition (EMT), and from endothelial to mesenchymal transition (EnMT). Myofibroblast differentiation is an early event in the development of fibrosis. Myofibroblast-like cells express smooth muscle (SM) cytoskeletal markers (a-SMA in particular) and participate actively in the production of extracellular matrix.

[0090] As also used herein, the term "myofibroblast-like cells" relates to cells characterized by expression of one or more cytoskeletal markers including vinculin, F-actin filaments, vimentin, fibroblast surface proteins, as well as increased production of a-smooth muscle actin. These cells may be further characterized by expression and secretion of one or more cytokines including IL-6, IL-8, VEGF, CXCL5, SDF-1, MMP1, CXCL6, COL4A4, MMP13, CYP7B1, ADAMDEC1, SLC6A1, CXCL1, PF4V1, CXCL3, CH25H, SFRP2, DARC, HCK, ERC2, CLIC6, BCL8 and combinations thereof.

[0091] The terms "patient", "subject", "host" or "individual" used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates {e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca {e.g., cynomologus monkeys such as Macaca fascicularis, and/or rhesus monkeys Macaca mulatta)) and baboon {Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees {Pan troglodytes)^'), rodents {e.g., mice, rats, guinea pigs), lagomorphs {e.g., rabbits, hares), bovines {e.g., cattle), ovines {e.g., sheep), caprines {e.g., goats), porcines {e.g., pigs), equines {e.g., horses), canines {e.g., dogs), felines {e.g., cats), avians {e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars), marine mammals {e.g., dolphins, whales), reptiles {e.g., snakes, frogs, lizards), and fish. In specific embodiments, the subject is a primate such as a human. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0092] As used herein, the terms "prevent," "prevented," or "preventing," refer to a prophylactic treatment which increases the resistance of a subject to developing the disease or condition or, in other words, decreases the likelihood that the subject will develop the disease or condition as well as a treatment after the disease or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse. These terms also include within their scope preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it.

[0093] "Protein expression" refers to transcription of a coding sequence encoded by a gene into messenger RNA (mRNA) and translation of the mRNA into protein. Herein, a sample or cell that "expresses" a protein of interest {e.g., a cell surface antigen such as ICAM-1) is one in which mRNA encoding the protein, or the protein, including fragments thereof, is determined to be present in the sample or cell.

[0094] The term "receptor" as used herein refers to a protein normally found on the surface of a cell {e.g., ICAM-1) which, when activated, leads to a signaling cascade in the cell.

[0095] The term "selective" refers to compounds that inhibit or display antagonism towards a molecule of interest without displaying substantial inhibition or antagonism towards another molecule. Accordingly, an agent that is selective for a target molecule inhibits or antagonizes that molecule more than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or more than about 100-fold, higher than it inhibits or antagonizes a non-target molecule.

[0096] As used herein a "small molecule" refers to a composition that has a molecular weight of less than 3 kilodaltons (kDa), and typically less than 1.5 kilodaltons, and more preferably less than about 1 kilodalton. Small molecules may be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules. As those skilled in the art will appreciate, based on the present description, extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, may be screened with any of the assays of the invention to identify compounds that modulate a bioactivity. A "small organic molecule" is an organic compound (or organic compound complexed with an inorganic compound {e.g., metal)) that has a molecular weight of less than 3 kilodaltons, less than 1.5 kilodaltons, or even less than about 1 kDa.

[0097] The term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0098] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base {e.g., A, T, C, G, I) or the identical amino acid residue {e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison {i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[0099] "Similarity" refers to the percentage number of amino acids that are identical or constitute conservative substitutions as suitably defined herein. Similarity may be determined using sequence comparison programs such as GAP (Deveraux eta/. 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0100] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence {i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually 50 to 100, more usually 100 to 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions {i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment {i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul eta/., 1997, Nucl . Acids Res. 25 : 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel eta/., "Current Protocols in Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter 15.

[OlOl] As used herein, the terms "treatment," "treating," and the like, refer to administering an agent, or carrying out a procedure {e.g., radiation, a surgical procedure, etc.) to obtain a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease. The effect may be therapeutic in terms of a partial or complete cure for a disease or condition {e.g., a chronic liver disease) and/or adverse effect attributable to the disease or condition. These terms also cover any treatment of a condition or disease in a mammal, particularly in a human, and include: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it {e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; (c) relieving the disease, i.e., causing regression of the disease; (d) reducing the severity of a symptom of the disease and/or (e) reducing the frequency of a symptom of the disease or condition.

[0102] By "vector" is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector may contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an

extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In the present case, the vector is suitably a viral or viral-derived vector, which is operably functional in animal and suitably mammalian cells. Such vector may be derived from a poxvirus, an adenovirus or yeast. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin) and the iiph gene which confers resistance to the antibiotic hygromycin B.

[0103] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

2. Methods of reducing H-Ferritin internalization

[0104] The present invention is based at least in part on the surprising finding that ICAM-1 is a major H-Ferritin receptor on HSCs, and is therefore an essential component in the H-Ferritin mediated proinflammatory signaling pathway. Specifically, the present inventors have found that H-Ferritin binds directly to ICAM-1, and therefore consider that inhibiting this interaction would inhibit internalization of H-Ferritin into the cell and/or inhibit inflammasome assembly, thereby reducing inflammation.

[0105] Based on these observations, the present inventors propose that a reduction of H-Ferritin internalization into cells that comprise a surface ICAM-1 polypeptide could be achieved by contacting the cell with an ICAM-1 antagonist. Thus, disclosed herein are methods of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, the methods comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, to thereby inhibit internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell . In some embodiments, the ICAM-1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, as set forth below: QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCP DGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANLTWLLRGEKELKREPAVGEP AEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTW CSLDG LFPVSEAQVH LALGDQRLN PTVTYG N DS FS AKASVS VTAE D EGTQ RLTCAVI LG N QSQETLQT VTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSAT LEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIG ESVTvTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEIVIITWAAAVIMGTAGLSTYLYNRQRKIKKYR LQQAQKGTPMKPNTQATPP [SEQ ID NO: 1] .

[0106] The present invention further encapsulates a method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 1 (Dl) {e.g., residues 1 to 83; or residues 1 to 84, as disclosed by Bella et a/., 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell . By way of an illustration, the ICAM-1 antagonists may be selected from the commercially available mAbs LAC1555, LAC3040, LAC3041, and LAC3043 (each of which is described in International Patent Publication No. WO 2005/086568, the entire contents of which is incorporated by reference in its entirety).

Alternatively, the ICAM-1 antagonist may be an antigen-binding molecule that competes for binding to Dl of an ICAM-1 polypeptide with any of these commercially available antibodies.

[0107] The invention also provides a method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 2 (D2) {e.g., residues 84 to 185; or residues 85 to 185, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell . By way of an example, the ICAM-1 antagonist may be the commercially available mAb, enlimomab.

Alternatively, the ICAM-1 antagonist may be an antigen-binding molecule that competes for binding to D2 of an ICAM-1 polypeptide with enlimomab.

[0108] The present invention also provides a method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 3 (D3) {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell . By way of an example, the ICAM-1 antagonist may be the mAb CBRICl/11 (as described in Parkos eta/., Mol Med, 1996, 2 : 489-505). Alternatively, the ICAM-1 antagonist may be an antigen-binding molecule that competes for binding to D3 of an ICAM-1 polypeptide with CBRICl/11. [0109] The present invention also provides a method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 4 (D4) {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell . By way of an example, the ICAM-1 antagonist may be the mAb CL203 (as described in Staunton eta/., Cell, 1990, 61 : 243-254). Alternatively, the ICAM-1 antagonist may be an antigen-binding molecule that competes for binding to D4 of an ICAM-1 polypeptide with CL203.

[OllO] Additionally, the present invention also provides a method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, and/or inhibiting inflammasome assembly in the cell, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 5 {e.g., residues 367 to 450; or residues 386 to 453, as disclosed by Bella etal, 1998) of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell, and/or inhibiting inflammasome assembly in the cell . By way of an example, the ICAM-1 antagonist may be the mAb CA-7 (as described in Rothlein etal, J Immunol, 1991, 147: 3788-3793). Alternatively, the ICAM-1 antagonist may be an antigen-binding molecule that competes for binding to D5 of an ICAM-1 polypeptide with CA-7.

[Olll] ICAM-1 is a transmembrane protein, and is a member of the immunoglobulin (Ig)-like superfamily. The full length polypeptide comprises five Ig-like domains, a transmembrane region, and a cytoplasmic tail of 28 amino acids. Both Dl and D3 of ICAM-1 are known to be essential for functional interactions with leukocyte β₂ integrins. For example, LFA-1 is known to bind Dl of ICAM-1, while MAC-1 binds D3. D4 and D5 of ICAM-1 (proximal to the membrane) forms a rigid stem structure.

[0112] ICAM-1 is known to be presented on the cell surface of a number of cells including HSCs, myofibroblasts or myofibroblast-like cells {e.g., liver myofibroblasts), endothelial cells, lymphocytes, and monocytes. In some preferred embodiments, the cell that comprises a cell surface ICAM-1 polypeptide is an HSC, or a myofibroblast or myofibroblast-like cell. In some of the same embodiments and other embodiments, the cells are other than Kupffer cells and hepatocytes.

2.1 ICAM-1 antagonists.

[0113] The ICAM-1 antagonist includes any molecule or compound that directly or indirectly binds or physically associates with an ICAM-1 polypeptide and that suitably blocks, inhibits, or otherwise antagonizes the binding of the ICAM-1 polypeptide to an H-Ferritin polypeptide. The binding or association may involve the formation of an induced magnetic field or paramagnetic field, covalent bond formation, ionic interaction (such as, for example, as occurs in an ionic lattice), a hydrogen bond, or alternatively, a van der Waals interaction such as, for example, a dipole-dipole interaction, dipole-induced dipole interaction, induced dipole-induced dipole interaction, or a repulsive interaction, or any combination of the above forces of attraction.

Accordingly, ICAM-1 antagonists for may be selected from peptides, peptidomimetics,

polypeptides, proteins, antigen-binding molecules, proteoglycans, nucleic acids, carbohydrates, sugars, lipids, small organic molecules, or other organic (carbon containing) or inorganic molecules, including those further described herein . In preferred embodiments, the ICAM-1 antagonist is a selective ICAM-1 antagonist.

2.1.1 Peptide/polypeptide antagonists.

[0114] The present invention further contemplates peptide or polypeptide based ICAM-1 antagonists that bind to a specific region of an ICAM-1 polypeptide, and preferably bind to the H- Ferritin-binding site on the ICAM-1 polypeptide. Suitable peptides of this type include the cyclic peptide inhibitors disclosed in International Patent Publication No. WO 2001/051508, the entire contents of which is incorporated herein by reference. The cyclic peptides taught therein are based around the amino acid sequence Cys-Leu-Leu-Arg-Met-Arg-Ser-Ile-Cys, with various amino acid substitutions and/or truncations also described that retain binding activity to an ICAM-1 polypeptide.

[0115] In some embodiments, the ICAM-1 antagonist comprises a peptide that comprises, consists, or consists essentially of an amino acid sequence of, or corresponding to, an H-Ferritin amino acid sequence, or a fragment thereof. Suitably, the ICAM-1 antagonist comprises at least about 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% similarity or identity to the naturally-occurring human H-Ferritin amino acid sequence.

Furthermore, the fragment of the variant H-Ferritin should be of sufficient length {e.g., at least about four amino acid residues) to bind an ICAM-1 polypeptide, and thereby prevent binding between an ICAM-1 polypeptide and an H-Ferritin polypeptide.

[0116] In certain embodiments of this type, the amino acid sequence of the peptide differs from the H-Ferritin amino acid sequence (or fragment thereof) by at least one amino acid substitution, addition, and/or deletion, such that the modified peptide retains the ability to bind the ICAM-1 polypeptide, but does not initiate a biological response or activity. Substituted amino acids may include conservative amino acid substitutions. Non-conservative substitutions may be tolerated, depending on the location of the substituted residues in the peptide, and other factors known to those skilled in the art. Exemplary conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

TABLE 1

AMINO ACID SUB-CLASSIFICATION

Residues that influence Glycine and Proline

chain orientation

[0117] Conservative amino acid substitutions also include groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in Table 2 under the heading of exemplary and preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bul k of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

TABLE 2

EXEMPLARY AND PREFERRED AMINO ACID SUBSTITUTIONS

Pro Gly Gly

Ser Thr Thr

Thr Ser Ser

Trp Tyr Tyr

Tyr Trp, Phe, Thr, Ser Phe

Val He, Leu, Met, Phe, Ala, Leu

Norleu

[0118] Alternatively or in addition, substituted amino acids or added amino acids can be any non-naturally occurring amino acids or derivatives thereof. Non-naturally occurring amino acids include chemical analogues of a corresponding naturally occurring amino acid. Examples of unnatural amino acids and derivatives include, bur are not limited to, 4-amino butyric acid, 6- aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6- methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2- thienyl alanine and/or D-isomers of amino acids.

[0119] In some embodiments, the peptide that comprises, consists, or consists essentially of an amino acid sequence corresponding to the naturally-occurring human H-Ferritin sequence as set forth in SEQ ID NO: 2, or a biologically active fragment thereof, or a variant of the naturally-occurring human H-Ferritin sequence, or a biologically active fragment thereof, is or is a derivative of a homologue or isoform of the naturally-occurring human H-Ferritin sequence. A "homologue" is a molecule from a different species and which is related by descent from a common ancestral DNA sequence. The term "homologue" may apply to the relationship between genes separated by the event of speciation or to the relationship between genes separated by the event of genetic duplication. An "isoform" is a peptide that has the same function as another peptide but which is encoded by a different polynucleotide and may have small differences in its sequence.

[0120] Peptides suitable for use in the present invention may be prepared in recombinant form using standard protocols as, for example, described in Sambrook eta/.,

MOLECULAR CLONING, A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), in particular Sections 16 and 17; Ausubel etal., CURRENT PROTOCOLS IN COLECULE BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan etal., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5, and 6. Typically, the peptide may be prepared by a procedure including the steps of (1) providing an expression vector from which the peptide is expressible; (b) introducing the vector into a suitable host cell; (c) culturing the host cell to express recombinant peptide from the vector; and (d) isolating the recombinant peptide. Alternatively, the peptide can be synthesized using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1998) or by Roberge etal, (1995, Science, 269 : 202). Syntheses may employ, for example, either f-butyloxycarbonyl (t-Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) chemistries {see, Chapter 9.1 of Coligan etal, supra; Stewart and Young, 1984, SOLID PHASE PEPTIDE SYNTHESIS, 2^nd' Pierce Chemical Co, Rockford, 111, 1994). 2.1.2 Antigen-binding molecules.

[0121] In some preferred embodiments the ICAM-1 antagonist is an anti-ICAM-1 antigen binding molecule. An "anti-ICAM-1 antigen-binding molecule" refers to an antigen-binding molecule that binds specifically to ICAM-1, for example recognizes and binds to a specific region of an ICAM-1 polypeptide and does not substantially bind to any other ICAM isoform {e.g., ICAM-2 ICAM-3, ICAM-4, or ICAM-5). In some embodiments, the anti-ICAM-1 antigen-binding molecule is a monoclonal antibody (mAb). Examples of some commercially available anti-ICAM-1 mAbs include, but are not limited to, enlimomab (R6-5-D6; ATCC 9580), YN l/1 (ATCC CRL-1878), and MD-2 (9F1B1). Antigen-binding molecules also encompass antigen-binding fragments of full-length anti-ICAM-1 mAbs, which are also suitable in the methods and compositions of the present invention. An antigen-binding fragment of a full-length anti-ICAM-1 mAb substantially retains activity of the complete antibody {e.g., the antigen-binding fragment blocks or inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide). Anti-ICAM-1 antigen- binding molecules can be made and used by those of ordinary skill in the art without undue experimentation {see, for example U.S. Patent No. 5,284,931).

[0122] In some embodiments, the ICAM-1 antagonist is an anti-ICAM-1 antigen-binding molecule that binds specifically to a region within Dl of ICAM-1. Dl may comprise amino acid residues 1 to 83; or residues 1 to 84, as disclosed by Bella eta/., 1998, of the mature ICAM-1 polypeptide sequence set forth in SEQ ID NO: 1. Representative anti-ICAM-1 antigen-binding molecules that specifically bind Dl of ICAM-1 include the mAbs LAC1555, LAC3041, and LAC3043 (each of which is described in International Patent Publication No. WO 2005/086568). Alternatively, the anti-ICAM-1 antigen-binding molecule may compete with any one of these antibodies for binding to Dl of an ICAM-1 polypeptide.

[0123] In other embodiments, the ICAM-1 antagonist is an anti-ICAM-1 antigen-binding molecule that specifically binds to a region within D2 of ICAM-1. D2 may comprise amino acid residues 84 to 185, or residues 85 to 185, as disclosed by Bella eta/., 1998, of the mature ICAM-1 polypeptide sequence set forth in SEQ ID NO: 1. Representative anti-ICAM-1 antigen-binding molecules that specifically bind D2 of ICAM-1 include the mAb enlimomab. Alternatively, the anti- ICAM-1 antigen-binding molecule may compete with enlimomab for binding to a region of D2 of an ICAM-1 polypeptide.

[0124] In still other embodiments, the ICAM-1 antagonist is an anti-ICAM-1 antigen- binding molecule that specifically binds to a region within D3 of ICAM-1. D3 may comprise amino acid residues 186 to 281, or residues 186 to 284, as disclosed by Bella eta/., 1998, of the mature ICAM-1 polypeptide sequence set forth in SEQ ID NO: 1. By way of an example, the anti-ICAM-1 antigen-binding molecule may be the mAb CBRICl/11 (as described in Parkos eta/., Mo/ Med, 1996, 2 : 489-505). Alternatively, the anti-ICAM-1 antigen-binding molecule may compete with CBRICl/11 for binding to a region of D3 of an ICAM-1 polypeptide.

[0125] In still other embodiments, the ICAM-1 antagonist is an anti-ICAM-1 antigen- binding molecule that specifically binds to a region within D4 of ICAM-1. D4 my comprise amino acid residues 282 to 366, or residues 285 to 385, as disclosed by Bella eta/., 1998, of the mature ICAM-1 polypeptide sequence set forth in SEQ ID NO: 1. By way of an example, the anti-ICAM-1 antigen-binding molecule may be the mAb CL203 (as described in Staunton eta/., Cell, 1990, 61 : 243-254). Alternatively, the anti-ICAM-1 antigen-binding molecule may compete with CL203 for binding to a region of D4 of an ICAM-1 polypeptide. [0126] In further embodiments, the ICAM-1 antagonist is an antigen-binding molecule that specifically binds to a region within D5 of ICAM-1. D5 may comprise amino acid residues 367 to 450, or residues 386 to 453, as disclosed by Bella eta/., 1998, of the mature ICAM-1 polypeptide sequence set forth in SEQ ID NO: 1. By way of an example, the ICAM-1 antagonist may be the mAb CA-7 (as described in Rothlein eta/., J Immunol, 1991, 147: 3788-3793).

Alternatively, the anti-ICAM-1 antigen-binding molecule may compete with CA-7for binding to a region of D5 of an ICAM-1 polypeptide.

[0127] Anti-ICAM-1 antigen-binding molecules of the invention may have specificity for a region of the ICAM-1 polypeptide that differs from the region to which other known anti-ICAM-1 antigen-binding molecules bind. Competition assays can be employed to confirm such differences in specificity, wherein a lack of competition is evidence that the antigen-binding molecule is binding to a different region of the ICAM-1 polypeptide. By way of an illustrative example, it has previously been shown that enlimomab does not compete with any of LAC1555, LAC3041, and LAC3043 for binding to an ICAM-1 polypeptide.

[0128] The region to which the antigen-binding molecules bind may be linear {i.e., a consecutive stretch of amino acids) or discontinuous {i.e., multiple stretches of amino acids). In order to determine whether the region to which an antigen-binding protein binds is linear or discontinuous, analysis can be conducted with respect to the ability of the antigen-binding molecule to retain the ability to specifically bind overlapping peptides {e.g., 13-mer peptides with an overlap of 11 amino acids) covering different domains of ICAM-1 {e.g., domains 1 and 2). In this respect, in some embodiments the antigen-binding molecules encapsulated by the invention suitably bind a region located on two (or more) domains of an ICAM-1 polypeptide. By way of an illustrative example, the antigen-binding molecule LAC3040 (as described in WO2005/086568, supra) is known to specifically bind regions located in both Dl and D2 of an ICAM-1 polypeptide.

[0129] Generally, an antigen-binding molecule binds to a region of the ICAM-1 polypeptide with an affinity (i.e., measured by dissociation constant, KD) of less than about 100 nM, more preferably less than about 75 nM, and still more preferably less than about 30 nM.

Further preferred antigen-binding molecules specifically bind a region of an ICAM-1 polypeptide with an affinity of less than about 10 nM, and more preferably less than about 3 nM.

[0130] In some embodiments, the antigen-binding molecule is a monoclonal antibody

(mAb). Monoclonal antibodies are large complex molecules typically with a molecular weight of about 150,000 kDa. Generally, a natural antibody molecule contains two identical pairs of polypeptide chains, with each pair comprising one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable ("V") region involved in binding the target antigen, and a constant ("C") region that interacts with other components of the immune system. The light and heavy chain variable regions align with one another in three-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging about ten amino acids in length) called the complementarity determining regions ("CDRs"). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in three-dimensional space to form the actual antibody binding site which locks onto the target antigen. The position and length of the CDRs have been precisely defined (typically by the "Kabat" denotation system described in Kabat, E. eta/., Sequences of Proteins of

Immunological Interest, U.S. Department of Health and Human Services, 1983; 1987; but can alternatively be as defined by Chothia or a composite of Kabat and Chothia definitions). The part of a variable region not contained in the CDRs is called the framework (FR), which forms the environment for the CDRs.

[0131] In some embodiments, the anti-ICAM-1 antigen-binding domain is a humanized antibody. Humanized antibodies have CDRs from a donor antibody and variable region framework regions (FR) and constant regions from a human antibody. A typical humanized antibody comprises (i) a light chain comprising three CDRs from a mouse antibody {e.g., MD-2) a variable region framework from a human antibody (which can be, for example, from a mature human antibody, human germline sequence, composite of two or more human antibody sequences, or consensus of human antibody sequences), and a human constant region, and (ii) a heavy chain comprising three CDRs from a mouse antibody {e.g., MD-2), a variable region framework from a human antibody and a human constant region. The variable region frameworks can also include back mutations at a few (usually fewer than 1, 2,3, 4, 5 or 10) selected positions in which a human residue is replaced with the residue occupying the corresponding position of the mouse antibody {see, Queen eta/., US Patent Nos. 5,530,101 and 5,585,089) . Specifically, the amino acids to be replaced in the framework are generally chosen on the basis of their ability to interact with the CDRs. For example, the replaced amino acids can be adjacent to a CDR in the donor antibody sequence or within 4-6 angstroms of a CDR in the humanized antibody as measured in 3-dimensional space.

[0132] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Numerous antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with for binding {see, Padlan eta/., FASEB Journal '9 : 133-139 (1995); Vajdos eta/., J Mo/ Biol, 320 : 415-428 (2002); Iwahashi eta/., Mo/. Immunol. 36: 1079-1091, (1999); Tamura eta/., J Immunol, 2000, 164: 1432-1441 (2000)). The substitution of certain regions within CDRs is based on the same principle as omitting dispensable CDRs, namely that only a small subset of CDR residues, the CDRs, actually contact antigen. CDR residues not contacting the cognate antigen can be identified based on previous studies (for example, residues in CDRH2 are often not required), from regions of Kabat CDRs lying outside Chothia CDRs, by molecular modelling and/or empirically. Omitted CDRs or at least a residue thereof are usually substituted with an amino acid occupying the corresponding position in human acceptor sequence supplying the variable region framework sequences. The number of such substitutions to include reflects a balance of competing considerations. Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity. However, substitutions can also cause changes of affinity, and significant reductions in affinity are preferably avoided. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non- conservative substitutions. However, in general empirical substitutions do not have the advantage of mouse to human substitutions in reducing immunogenicity. Empirical substitutions can increase or decrease affinity of the resulting humanized antibody.

[0133] In general, humanized antibodies with a satisfactory binding affinity to ICAM-1 polypeptide and lack of substantial immunogenicity can be obtained by individual screening of a number of variants made according to the above principles above. However, very large numbers of variants can be simultaneously screened using a display selection method such as phage display {see, International PCT Publication Nos. W091/17271, WO92/001047, and WO92/20791). [0134] In some embodiments, the anti-ICAM-1 antigen-binding molecule is a chimeric antibody. Typically, chimeric antibodies comprise the heavy and light chain variable regions of a mouse (or other rodent) antibody, combined with the heavy and light chain constant regions of a human antibody. The construction of such chimeric antibodies by means of genetic engineering is well known in the art. Such antibodies retain the binding specificity of the mouse antibody, while being about two thirds human. The proportion of non-human sequence present in chimeric and humanized antibodies generally suggests that the immunogenicity of chimeric antibodies is intermediate between fully mouse and fully humanized antibodies.

[0135] Usually, humanized and chimeric antibodies are of the IgGl, IgG2, IgG3 or IgG4 isotype with a kappa (κ) light chain. In some preferred embodiments, the antigen-binding fragment is of the IgGl isotype.

[0136] The antigen-binding fragments of the invention also include binding fragments of antibodies such as Fv, Fab and F(ab')2; Afunctional hybrid antibodies {e.g., Lanzavecchia eta/., Eur. J. Immunol. 17: 105, 1987), single-chain antibodies {see, Huston eta/., Proc. Natl. Acad. Sci. USA 85 : 5879, 1988; Bird eta/., Science 242 : 423, 1988); and antibodies with altered constant regions {see, U.S. Patent No. 5,624,821).

3. Methods of reducing inflammation

[0137] Having regard to the ability of the ICAM-1 antagonists to inhibit inflammasome assembly, the present inventor also deduced that these molecules have substantial utility in methods of reducing inflammation {e.g., hepatic inflammation) in a subject. These methods generally comprise contacting a cell of the subject that comprises a cell surface ICAM-1 polypeptide with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H- Ferritin polypeptide, and thereby reducing inflammation in the subject.

[0138] More specifically, the present invention provides a method of reducing inflammation in a subject, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 1 {e.g., residues 1 to 83; or residues 1 to 84, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, to thereby reduce inflammation in the subject. Suitable ICAM-1 antagonists may be selected from the commercially available mAbs LAC1555, LAC3040, LAC3041, and

LAC3043 (each of which is described in International Patent Publication No. WO 2005/086568). Alternatively, the ICAM-1 antagonist may be an antigen-binding molecule that competes for binding to Dl of an ICAM-1 polypeptide with any of these commercially available antibodies.

[0139] In an alternative aspect, the invention provides a method of reducing inflammation in a subject, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 2 {e.g., residues 84 to 185; or residues 85 to 185, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby reducing inflammation in the subject. By way of an example, the ICAM-1 antagonist may be enlimomab. Alternatively, the ICAM-1 antagonist may be an antigen- binding molecule that competes for binding to D2 of an ICAM-1 polypeptide with enlimomab.

[0140] Yet another aspect of the invention is a method of reducing inflammation in a subject, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 3 {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby reducing inflammation in the subject. By way of an example, the ICAM-1 antagonist may be the mAb CBRICl/11 (as described in Parkos eta/., Mo/ Med, 1996, 2 : 489-505). Alternatively, the ICAM-1 antagonist may be an antigen-binding molecule that competes for binding to D3 of an ICAM-1 polypeptide with CBRICl/11.

[0141] The present invention also encapsulates a method of reducing inflammation in a subject, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 4 {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, thereby reducing inflammation in the subject. By way of an example, the ICAM-1 antagonist may be the mAb CL203 (as described in Staunton eta/., Cell, 1990, 61 : 243-254). Alternatively, the ICAM-1 antagonist may be an antigen-binding molecule that competes for binding to D4 of an ICAM-1 polypeptide with CL203.

[0142] Alternatively, the method of reducing inflammation in a subject, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 5 {e.g., residues 367 to 450; or residues 386 to 453, as disclosed by Bella etal, 1998) of the ICAM-1 polypeptide, reducing inflammation in the subject. By way of an example, the ICAM-1 antagonist may be the mAb CA-7 (as described in Rothlein etal, J Immunol, 1991, 147: 3788-3793). Alternatively, the ICAM-1 antagonist may be an antigen-binding molecule that competes for binding to D5 of an ICAM-1 polypeptide with CA-7.

[0143] Optionally, the methods of the present invention include a first step of identifying a subject in need of a prevention or reduction in inflammation {e.g., a subject suffering with a chronic liver disease).

[0144] The subject may or may not have been diagnosed with an inflammatory condition or a disease or condition that is associated with or causes an inflammatory condition. In some embodiments the inflammatory condition is a chronic liver disease such as fibrosis or cirrhosis. In some embodiments, the subject is not suffering from a chronic liver disease. In some embodiments, the inflammatory disease or condition is a carcinoma {e.g., HCC).

[0145] In some embodiments, the inflammation in the subject is localized to a particular location or tissue. By way of an illustration, fibrosis can occur in many tissues within a subject {e.g., liver, lungs, heart, brain, etc.) typically as a result of localized inflammation or damage. In some preferred embodiments the inflammation is localized to the liver tissue of the subject.

[0146] In related embodiments, a method of treating a chronic liver disease is encapsulated by the present invention . In methods of this type, the subject has a chronic liver disease such as fibrosis and/or cirrhosis. In one aspect, the fibrosis is located in the liver, which may include one or more of the hepatic ducts. Liver fibrosis is caused by proliferation of tough fibrous connective tissue in the liver. Common causes of liver or hepatic fibrosis include chronic infection by hepatitis B or hepatitis C, viruses, the parasite Schistosoma, chronic alcoholism, exposure to certain drugs and toxins, infections, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), inherited metabolic diseases like hemochromatosis, Wilson's disease, a-1 antitrypsin deficiency, chronic liver disease, autoimmune diseases such as primary biliary cirrhosis and auto-immune hepatitis, genetic predisposition to fibrosis, or complications from secondary illnesses, including the fibrosis of other organs or auto-immune disorders. In one aspect, the fibrosis is idiopathic.

4. Methods of producing anti-inflammatory agents

[0147] The present invention also provides methods for identifying agents that inhibit the binding of an ICAM-1 polypeptide with an H-Ferritin polypeptide, and methods of producing anti-inflammatory agents incorporating these identification methods. The methods may comprise screening for an agent that inhibits the binding of an ICAM-1 polypeptide and an H-Ferritin polypeptide. In its broadest form, illustrative methods may comprise contacting a cell that comprises an ICAM-1 polypeptide on its cell surface with a candidate agent and an H-Ferritin polypeptide. The level of binding between the ICAM-1 polypeptide and the H-Ferritin polypeptide is measured directly {e.g., binding assay) or indirectly (downstream function or activity assay). A detected decrease in the level of binding between the ICAM-1 polypeptide and the H-Ferritin polypeptide relative to the level of binding in the absence of the candidate agent, indicates that the agent is useful as an anti-inflammatory agent.

[0148] In some embodiments, the methods of producing an anti-inflammatory agent comprise: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to Dl {e.g., residues 1 to 83; or residues 1 to 84, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0149] In other embodiments, the methods of producing an anti-inflammatory agent comprise: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to D2 {e.g., residues 84 to 185; or residues 85 to 185, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0150] In other embodiments, the methods of producing an anti-inflammatory agent comprise: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to D3 {e.g., residues 186 to 281; or residues 186 to 284, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0151] In other embodiments, the methods of producing an anti-inflammatory agent comprise: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate

ICAM-1 antagonist that specifically binds to D4 {e.g., residues 282 to 366; or residues 285 to 385, as disclosed by Bella eta/., 1998) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0152] In other embodiments, the methods of producing an anti-inflammatory agent comprise: (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate that specifically binds to D5 (residues 387 to 450) of the ICAM-1 polypeptide, in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an antiinflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

[0153] As such, the present invention provides methods of identifying an agent for use in the prevention of inflammation {e.g., hepatic inflammation). These methods generally comprise determining whether a candidate agent is capable of antagonizing the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide. For example the methods may comprise determining whether a candidate agent is capable of decreasing the amount of H-Ferritin internalization into a cell that comprises an ICAM-1 polypeptide on its surface.

[0154] A candidate agent for use in the production methods include to any compound, molecule, or agent that may potentially antagonize ICAM-1. The candidate agent may be, or may comprise, for example, a peptide, polypeptide, protein, antibody, polynucleotide, small organic molecule or other compound, for example, designed through rational drug design starting from known antagonists of ICAM-1.

[0155] The candidate agent could be derived or synthesized from chemical compositions or man-made compounds. Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Suitable candidate agents which can be tested in the above assays include compounds from combinatorial libraries, small molecule libraries, and natural product libraries, such as display {e.g., phage display) libraries. Multiple candidate agents may be assessed using a method of the invention in order to identify one or more agents having a suitable effect on an ICAM-1 polypeptide, such as a reduction its binding to H- Ferritin.

[0156] The contacting and detecting steps of the methods may be carried out in vivo, ex vivo, or in vitro. In particular, the step of contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate agent in the presence of H-Ferritin may be carried out in vivo, ex vivo, or in vitro. The ability of a candidate agent to inhibit the binding of an ICAM-1 polypeptide with an H-Ferritin polypeptide may be tested in any cell that comprises a cell surface ICAM-1 polypeptide. For example, the cell may be an HSC, or a myofibroblast or myofibroblast-like cell {e.g., a liver myofibroblast). In preferred embodiment, the cell is other than a hepatocyte or Kupffer cell.

[0157] A variety of techniques for measuring polypeptide ligand binding to a cognate receptor are known in the art, and any suitable method for detecting a reduction in binding of an ICAM-1 polypeptide to an H-Ferritin polypeptide, including those assays that measure an output from the cells that comprise an ICAM-1 polypeptide on its cell surface. This output may be the production of an inflammatory cytokine by the cell {e.g., HSC, or a myofibroblast or myofibroblast- like cell, inclusive of a liver myofibroblast). For example, the IL-1 family consists of 11 members, seven of which have possess broad pro-inflammatory activity {i.e., IL-la, IL-Ιβ, IL-18, IL-33, IL- 36a, ΙΙ_-36β and IL-36y), and one comprises anti-inflammatory {i.e., IL-37) activity. By way of an illustration, when the cells are exposed to a candidate agent, if the candidate agent is an anti- inflammatory agent it will inhibit the binding of the ICAM-1 polypeptide to its ligand, H-Ferritin. This in turn, will result in a decrease in H-Ferritin polypeptide internalization into the cell and therefore a reduction in inflammasome assembly. Thus, the amount of IL-Ιβ (and other inflammatory cytokine) produced by the cell will be reduced . Accordingly, measurement of IL-Ιβ production following the addition of a candidate agent can be used to determine the anti- inflammatory potential of the candidate agent.

[0158] In other words, when the H-Ferritin polypeptide binds to the ICAM-1 polypeptide that is located on the surface of a cell, the cell is stimulated. This stimulation can be monitored by the production of cytokines and/or chemokines known to be secreted by said cell (for example, by an ELISPOT assay or Western blot). Thus, it is possible to detect ICAM-1 polypeptide binding to an H-Ferritin polypeptide by any of a number of techniques known in the art.

[0159] Any cytotoxicity assay is suitable for using to assess the ability of an ICAM-1 polypeptide to bind to an H-Ferritin polypeptide {see, Chapter 6.2 to 6.24 in Current Protocols in Immunology (1999), edited by Coligan J. E., Kruisbeek A. M., Margulies D. H., Shevach E. M. and Strober W., John Wiley & Sons). Other methods for screening immunogenic activity include techniques well known to the skilled artisan (for example, immunoassays such as ELISA). By way of example, such screening methods may be performed using methods such as those described in Harlow and Lane, Antibodies/ A Laboratory Manual, Cold Spring Harbour Laboratory, 1988. Altered levels of an inflammatory cytokines and/or chemokines {e.g., IL-1) is a clear indication that the candidate agent is a ICAM-1 antagonist that is suitable for use with the present invention (i .e., reduces binding of the ICAM-1 polypeptide with the H-Ferritin polypeptide).

[0160] Enzyme-linked immunosorbent assay (ELISA) has long been used to detect and measure cytokine and/or chemokine levels. ELISA is commonly used in the evaluation and characterization of immune responses, and methods for performing such analysis are well known in the art. In some embodiments, a multi-analyte ELISA assay may be performed, with allows for the rapid screening of the secretion of up to 12 cytokines and/or chemokines in a single experiment.

[0161] Variations of the traditional ELISA method, including ELISPOT assays, are also well known in the art.

[0162] The enzyme linked immunospot (ELISPOT) assay (such as described in European Patent No. EP0941478) can measure the activation of immobilized effector cells {e.g., a cell that comprises an ICAM-1 polypeptide on the cell surface) in a sample by detecting cytokines secreted by effector cells {e.g., the cell comprising an ICAM-1 polypeptide on the cell surface) in response to contact with an H-Ferritin polypeptide and the candidate agent, through capture of the cytokines and/or chemokines secreted by responding effector cells {e.g., the cell comprising an ICAM-1 polypeptide on the cell surface) in their vicinity on an adsorber membrane with a cytokine- and/or chemokine-specific antibody {e.g., monoclonal antibody). The cytokine capture is then detected with a second anti-cytokine antibody, which binds to a different epitope on the same cytokine compared to the capture antibody. Binding of the second antibody is then detected with a color- based labeling reaction. As a consequence, stimulated cells are then detected as colored spots on the membrane, which form around the original location where the cell was immobilized. [0163] This technique therefore enables the quantification of a specific secreting a specific cytokine and/or chemokine in response to an ICAM-1 antagonist. Basically, cytokine and/or chemokine secreting cells {e.g., hepatic stellate cells that comprise an ICAM-1 polypeptide on their cell surface) may be revealed by culturing the cells in specially modified ELISA wells that contain antibody to the cytokine and/or chemokine of interest bound to the well surface, together with the H-Ferritin polypeptide and candidate agent. In this method, the standard ELISA reagents are replaced with enzyme-substrate complexes that yield a colored precipitate (spots), adjacent to the secreting cell. Spots can then be counted to give a measure of the number of cytokine- and/or chemokine-producing cells.

[0164] Further advantage of performing ELISPOT assays is that advanced operator training and expensive equipment are not necessary for measuring frequency of responses. In some embodiments, ELISPOT evaluates IL-Ιβ production, which occurs in large quantities as this cytokine is considered to be prototypic of an inflammatory response. However, other cytokines can be assessed and the measurement of which is suitable for the present invention. ELISPOT analysis may be improved by adding a computer-assisted microscope to simplify the assay readout, and allows batch analysis of large series of samples, and facilitates standardization (as described in Samri eta/., 2006).

[0165] When the candidate agents have been screened, a rescreening process may occur in the same manner to confirm the identified activity. Additional assays may be performed with identified ICAM-1 antagonists if desired.

[0166] A candidate agent that identified as an anti-inflammatory agent may result in a decrease in the binding of an ICAM-1 polypeptide and an H-Ferritin polypeptide of at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 75%, or at least 85% or more in the presence of the candidate agent as compared to in the absence of the candidate agent. A candidate agent that is identified as an anti-inflammatory agent may result in a decrease in the binding of an ICAM-1 polypeptide to an H-Ferritin polypeptide such that binding is no longer detectable in the presence of the candidate agent. Such a decrease may be seen in the sample being tested or, for example, where the method is carried out in an animal model, in particular tissue from the animal such as in the circulation of other organs such as the liver.

4.1 Methods of producing antigen-binding molecules.

[0167] Antigen-binding molecule {e.g., mAbs) may be generated in response to immunization with fragments of ICAM-1 polypeptide. The antigen-binding molecules can then be assayed to identify those antibodies which are capable of inhibiting the ability of an ICAM-1 polypeptide to bind with an H-Ferritin polypeptide. These antigen-binding molecules may be used as anti-inflammatory agents, and thus will have utility in the methods disclosed above and elsewhere herein

[0168] Identified anti-inflammatory agents that are antigen-binding molecules may be of animal {e.g., mouse, rat, hamster or chicken) origin, or they may be genetically engineered. For example, rodent mAbs can be made by well-established methods in the art, comprising multiple immunizations with an ICAM-1 polypeptide in an appropriate adjuvant (either i.p., i.v., or into the footpad), followed by extraction of spleen or lymph node cells and fusion with a suitable immortalized cell line. Hybridomas that produce antibody binding to the ICAM-1 polypeptide are then selected. Human antibodies can also be made by phage display (see, International Patent

Publication Nos. W091/17271, WO92/001047, and WO92/20791, and Winter, FEBS Lett. 23 : 92, 1998) or by using transgenic mice {see, e.g., International Patent Publication Nos. W093/12227 and WO91/10741).

[0169] Native mAbs of the invention may be produced from their hybridomas.

Genetically engineered mAbs, e.g., chimeric or humanized mAbs, may be expressed by a variety of art-known methods. For example, genes encoding their light and heavy chain V regions may be synthesized from overlapping oligonucleotides and inserted together with available C regions into expression vectors {e.g., commercially available from Invitrogen) that provide the necessary regulatory regions, e.g., promoters, enhancers, poly A sites, etc. Use of the CMV promoter- enhancer is generally preferred. The expression vectors may then be transfected using various well-known methods such as lipofection or electroporation into a variety of mammalian cell lines such as CHO or non-producing myelomas including Sp2/0 and NSO, and cells expressing the antibodies were selected by appropriate antibiotic selection.

[0170] Once expressed, the antibodies {e.g., mAb) of the invention may be purified according to standard procedures of the art such as microfiltration, ultrafiltration, protein A or G affinity chromatography, size exclusion chromatography, anion exchange chromatography, cation exchange chromatography and/or other forms of affinity chromatography based on organic dyes or the like. Substantially pure antibody preparations of at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, or above 95% homogeneity are preferred. Furthermore, for pharmaceutical uses antibody preparations of at least about 96, 96, 98, 99, 99.5%, or higher homogeneity are most preferred.

S. Pharmaceutical compositions

[0171] In accordance with the present invention, it is proposed that ICAM-1 antagonists are useful for the prevention or treatment of inflammatory conditions in a subject {e.g., chronic liver diseases, such as fibrosis and cirrhosis, In some embodiments, the inflammatory disease or condition is a chronic liver disease, or a carcinoma {e.g., HCC)). In specific embodiments, the antagonists are administered to the subject after identifying that the subject has or is at risk of developing an acute inflammatory condition. In accordance with the present invention, the ICAM-1 antagonist may be any agent that inhibits the binding of an ICAM-1 polypeptide and an H-Ferritin polypeptide, including those described herein, and those ICAM-1 antagonists identified using the screening methods broadly described above.

[0172] The ICAM-1 antagonists can be administered to an individual either by themselves, or in pharmaceutical compositions where they are mixed with a suitable

pharmaceutically acceptable carrier or diluent.

[0173] The ICAM-1 antagonists of the present invention may be conjugated with biological targeting agents which enable their activity to be restricted to particular cell types. Such biological-targeting agents include substances which are immuno-interactive with cell-specific surface antigens.

[0174] The ICAM-1 antagonists may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's

Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the agents may be formulated in aqueous solutions, suitably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal

administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Intra-muscular and subcutaneous injection is also contemplated.

[0175] The ICAM-1 antagonists can be formulated readily using pharmaceutically acceptable carriers or diluents well known in the art into dosages suitable for oral administration. Such carriers or diluents enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers or diluents may be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen- free water.

[0176] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. The dose of agent administered to an individual should be sufficient to effect a beneficial response in the individual over time such as reducing or otherwise ameliorating in an individual the symptoms of an acute inflammatory condition or the symptoms of a disease or condition that causes or is otherwise associated with an acute inflammatory condition. The quantity of the agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the agent(s) for administration will depend on the judgment of the practitioner. In determining the effective amount of the drug to be administered, the physician may evaluate the characteristics of the patient, their response to the drug and the safety profile of the drug. In any event, those of skill in the art may readily determine suitable dosages of the agents.

[0177] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or other components which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0178] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid carriers, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable carriers are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more drugs as described above with the carrier or diluent which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0179] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0180] Pharmaceuticals which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol . The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

[0181] Dosage forms of the agents 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 an agent of the invention 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 hydroxypropyl methyl cellulose. In addition, controlled release may be effected by using other polymer matrices, liposomes or microspheres.

[0182] The ICAM-1 antagonists may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

[0183] For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC₅₀ as determined in cell culture {e.g., the concentration of a test agent, which achieves a half-maximal antagonism in activity of ICAM-1 ). Such information can be used to more accurately determine useful doses in humans.

[0184] Toxicity and therapeutic efficacy of such drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅o/ED₅o. Compounds that exhibit large therapeutic indices may be employed. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. In some embodiments the dosage of such compounds lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See for example Fingl et a/., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi).

[0185] Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain C3aR function agonistic or activation effects. Usual patient dosages for systemic administration range from 1-2000 mg/day, commonly from 1-250 mg/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02-3 mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5- 1200 mg/m2/day, commonly from 0.5-150 mg/m2/day, typically from 5-100 mg/m2/day.

[0186] Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a tissue, which may be subcutaneous or omental tissue, often in a depot or sustained release formulation.

[0187] Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the tissue.

[0188] In cases of local administration or selective uptake, the effective local concentration of the agent may not be related to plasma concentration.

6. Kits

[0189] The present invention also provides kits comprising an ICAM-1 antagonist or pharmaceutical composition as broadly described above and elsewhere herein. Such kits may additionally comprises additional immunomodulating agents for concurrent use with the ICAM-1 antagonists or pharmaceutical compositions of the invention.

[0190] The kit may comprise additional components to assist in performing the methods of the present invention such as, for example, administration device(s), buffer(s), and/or diluent(s). The kits may include containers for housing the various components and instructions for using the kit components in the methods of the present invention.

[0191] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples. EXAMPLES

EXAMPLE 1

H-FERRITIN DRIVES SECRETION OF IL-Ιβ

[0192] It was contemplated that in for H-Ferritin to contribute to liver inflammation it should be observed to stimulate activation and secretion of IL-Ιβ by HSC, a process that requires Caspase-1 activation by the inflammasome {see, Martinon et al. 2002). Figure 1 clearly demonstrates that in primary HSC, H-Ferritin (ΙΟηΜ, 24 hours) stimulates the expression of NLRP3 protein . Notably, no changes in NLRP1 is observed in either H-Ferritin-treated HSC {see, Figure IB). Moreover, as an evidence of IL-Ιβ processing, the present inventors observed H-Ferritin to increase the level of activated Caspasel {i.e., fragment of 20kDa) {see, Figure 1A) as well as of the inflammasome-adaptor protein Apoptosis-associated speck-like (ASC) {see, Figures 1C-D). Indeed, confocal imaging showed that ASC exhibited high levels of co-localization with IL-Ιβ in H-Ferritin- treated cells {see, Figure ID). LPS (lOOng/mL) was used as a positive control to show similar, but perhaps weaker co-localization of ASC and IL-Ιβ (Figure ID). In HSCs in which either NLRP3, NLRP1 or NLRC4 were knocked down, there was a decrease vin the expression of NLRP3 protein (Figure IE). In addition, there was an almost complete inhibition of IL-Ιβ protein secretion induced by H-Ferritin in these same cells where either NLRP3, NLRP1 or NLRC4 were knocked down (Figure IF), which demonstrated the role of H-Ferritin in the activation of the inflammasome.

[0193] In liver tissue, expression and secretion of cytokines and in particular of IL-Ιβ, can be originated in hepatocytes and Kupffer cells in addition to in HSC. However, liver Kupffer cells are unlikely to increase cytokine synthesis in response to H-Ferritin as our previous investigations have shown that Kupffer cells lack membrane-bound Ferritin receptors (Ramm GA et a/., JCI 1994).

[0194] As Kupffer cells have previously been shown to lack membrane-bound H-Ferritin receptors (see, Ramm eta/., JCI, 1994), it was considered unlikely that these cells increase cytokine synthesis in response to H-Ferritin. Furthermore, experiments in a hepatocarcinoma cell line Huh7 (to model hepatocytes) suggested that H-Ferritin (10 nM) does not stimulate IL-Ιβ expression in hepatocytes {see, Figure 1G). Thus, it was hypothesized that H-Ferritin induces the expression of IL-Ιβ specifically in HSCs. To assess whether H-Ferritin has the capacity to stimulate the expression of IL-Ιβ in liver tissue, ex vivo slices of mouse liver tissue were treated with H- Ferritin in comparison to LPS. Individual liver slices were incubated for 12 hours in the presence of H-Ferritin (10 nM) or LPS ( 100 ng/mL). Notably, it is observed that H-Ferritin stimulated IL-Ιβ expression to a similar extent as the LPS positive control (Figure 1H). Accordingly, the combination of H-Ferritin failing to have any stimulatory effect on IL-Ιβ in cultured Huh7 cells, and the lack binding to liver-derived Kupffer cells, it is demonstrated that a significant enhancement of IL-Ιβ expression might have originated in the resident HSC of the liver slices, rather than in Kupffer cells or in hepatocytes. These data provide the first evidence to support that H-Ferritin and HSC are the primary direct drivers of hepatic inflammation in vivo.

Materials and Methods

[0195] Primary Hepatic Stellate cell isolation. Studies were performed with institutional Animal Ethics Committee approval and compliance with Australian regulatory guidelines. Rat HSCs were isolated from normal male Sprague-Dawley rats (600 ± 50 g) by sequential pronase/collagenase perfusion, cultured on plastic 12-well plates and grown in Medium 199 (Invitrogen) supplemented with 10% calf + 10% horse serum, 50 pg/mL ascorbic acid, and penicillin/streptomycin (100 U and 100 pg/mL, respectively), to induce an activated phenotype. In addition, isolated HSCs were cultured on Teflon tissue culture inserts (Millipore) for 24 hours to maintain cells in a quiescent phenotype. Twenty-four hours before experimentation, HSCs were washed and cultured in serum-free M199 medium. HSCs were then treated with Recombinant human H-Ferritin (FTHl). Recombinant ferritins rHF and rLF were prepared as described in Santambrogio P et al., J Biol Chem 1993; 268: 12744-12748. Recombinant FLAG -FTHl

(Cat#RC209845) and FLAG-FTLl (L-Ferritin, Cat#RC203296) originated in human kidney HEK cells were purchased from Origen (also referred to as human generated FTH l and FTLl). Five-day culture-activated HSCs were routinely used except where indicated.

[0196] Extraction of RNA from HSC. Subconfluent (80%-90%) rat HSCs were washed with ice-cold phosphate-buffered saline and placed on ice before total RNA isolation using the RNeasy mini kit (Qiagen) as per the manufacturer's instructions. Total RNA was then treated with 1 μΙ_ RNA-qualified ribonuclease-free deoxyribonuclease I (ΐυ/μΙ_; Promega) per 1 pg total RNA for 30 minutes at 37°C to ensure complete removal of DNA contamination. Total RNA concentration and quality was estimated by spectrophotometry at 260 to 280 nm.

[0197] Quantitative Real-Time Reverse Transcription Polymerase Chain

Reaction. 1 pg was reverse-transcribed into cDNA using SensiFast (Bioline). Real-time polymerase chain reaction (PCR) reactions were performed in a final volume of 15 μΙ_ with forward and reverse oligonucleotide primers used at a final concentration of 500 nM, using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and the reaction profile as recommended by the manufacturer. All PCR reaction efficiencies were between 0.95 and 1.10. Melt curve analysis revealed the presence of a single product with a single melt signature. PCR was performed using an RG-6000 thermal cycler (Corbett Research Australia). Messenger RNA (mRNA) quantitation was achieved using the two standard curves method, with the fluorescence measurements of the unknown samples back- referenced to a standard curve relating concentration to fluorescence in arbitrary units. Messenger RNA levels were normalized to expression of the housekeeping gene Basic Transcription Factor 3 and expressed relative to untreated or control samples (presented as relative mRNA expression). IL-Ιβ (NM_031512) : Forward 5'-ATCCCAAACAATACCCAAAGAAGAA-3' and Reverse 5'- TG G G G AACTGTG CAG ACTCAAAC- 3 '; BTF3 (N M_001008309) : 5'-TGGCAGCAAACACCTTCACC-3' and Reverse 5'-AGCTTCAGCCAGTCTCCTCAAAC-3'; ICAM-1 (NM_012967) : 5'- TG ATCATTG CG G G CTTCGTG - 3 ' and reverse 5'-GGCGGGG CTTGTACCTTG AGT- 3 ';

[0198] RT-qPCR. Total RNA was extracted from both untreated (control) and taurocholate-treated PIL-2 cells using the RNeasy kit (Qiagen) and 1 pg was reverse-transcribed into cDNA using SensiFast (Bioline). Real-time qPCR to quantitate mRNA expression was performed on a Light Cycler Instrument (Roche Molecular Biochemicals) with BTF3 used as the reference gene.

[0199] Knocking-down experiments. For knocking down ICAM1 we pooled three different duplex targeting rat ICAM1 (NM_012967): SASI_Rn01_00094819;

SASI_Rn01_00094824 and SASI_Rn01_00094825; For knocking-down of rat NLRP1, NLRP3 and NLRC4 in rat HSC, specific siRNA were ordered to SIGMA-Aldrich and 5 nmol of each duplex is pooled into a single tube and the remaining 5 nmol of each individual siRNA is shipped in its own tube. The reference number for each siRNA is: Rat NLRP1 pool: SASI_Rn02_00391969;

SASI_Rn02_00391970; SASI_Rn02_00391971; SASI_Rn02_00391972. Rat NLRP3 pool:

SASI_Rn02_00349589; SASI_Rn02_00349590; SASI_Rn02_00349591 and SASI_Rn02_00349592. Rat NLRC4 pool: SASI_Rn02_00221598; SASI_Rn02_00221599; SASI_Rn02_00221600 and SASI_Rn02_00221601. siRNA were transfected twice as described above for cDNA ICAMl-GFP vector. HSCs were left 24 hours recovering in completed M 199 media between two transfections.

[0200] Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western blotting. Whole HSC cell protein extracts were prepared by lysis of HSC cultures in RIPA buffer and lx sodium dodecyl sulfate (SDS) sample buffer (62.5 rnM Tris HCI pH6.8, 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% wt/vol bromophenol blue). Twenty micrograms of cell protein was subjected to SDS polyacrylamide gel electrophoresis and western blot using specific antibodies. Western blots were probed with anti-ASC (Santa Cruz sc-514414; 1: 500), anti- Caspase-1 (Adipogen, AG-20B-0042-C100, 1 : 1000), anti-NLRP3 (Adipogen, AG-20B-0014;

1 : 1000), anti-NLRPl (Adipogen, AG-25B-0005, 1 : 1000) and anti^-actin (Cell Signaling; l : 1000)as a loading control in TBST with 5% BSA powder. Blots were probed with an anti-rabbit/goat/mouse secondary at 1 : 5000-10000 in TBST 5% skim milk powder. HPR localization was detected by Western Lightning Plus ECL (Perkin Elmer) and visualized using X-ray film.

[0201] Immunofluorescence. To examine rat HSC cells were grown on glass coverslips to and treated for 24hours with vehicle (PBS), FTHl (24hours, 10 nM) and LPS (24hours, 100 ng/mL). Cells were fixed with formalin, blocked with 0.2% BSA and probed with anti-ACS (Santa Cruz sc-514414; 1 : 250) or ΙΙΙβ (Santa Cruz sc-7884; 1 : 250) for lhour at room

temperature and then treated with Alexa Fluor 594 donkey anti-mouse and Alexa Fluor 488 donkey anti-rabbit (Invitrogen) at 1 : 500 for one hour, respectively. Glass coverslips were mounted in Prolong Gold (Molecular Probes) with DAPI. Images were taken with confocal microscope Zeiss 780NLO.

[0202] Ex vivo liver slice studies. Precision-cut (ex vivo) liver slices were prepared (minimum of three liver slices each) from three separate C57BL6 WT control mice for each time point and each treatment group, were incubated in M 199 medium at 37°C for. Slices were harvested for mRNA quantitation using RT-qPCR 12 hours later.

[0203] Quantification of IL-Ιβ protein secretion from HSCs. The concentrations of

Ιίΐβ released from HSC, were quantified using the ELISA assay (R&D Systems), according to the manufacturer's protocol. Concentrations were normalized as fold change in regarding FTHl-treated HSCs in each experimental replicate.

[0204] Statistical analysis. Results are presented as mean ± SEM . Statistical analysis was performed with GraphPad Prism 6 (GraphPad Software Inc.). Groups were compared using one-way analysis of variance (ANOVA) with Dunnett's multiple comparison post hoc test or the Student r test, where applicable using Prism 4.0 (GraphPad Software). P < 0.05 was considered statistically significant.

EXAMPLE 2

ICAM-1 is THE PRIMARY H-FERRITIN RECEPTOR RESPONSIBLE FOR IL-Ιβ EXPRESSION

[0205] Since H-Ferritin plays a crucial role in the development and progression of hepatic inflammation, novel strategies to modulate H-Ferritin signaling will be crucial for ameliorate the burden of liver disease. In order to achieve this, the molecules governing the ability of H-Ferritin to stimulate IL-Ιβ expression in HSC were elucidated. Previous studies have postulated that H-Ferritin receptors include T-cell immunoglobulin and mucin domain containing 2 (TIM-2), transferrin receptor-1 (TfRl), scavenger receptor class A member 5 (Scara5). However, none of these receptors have been shown by the present inventors to be involved in stimulating IL-Ιβ expression in HSCs (data not shown).

[0206] Accordingly, two different experimental approaches were adopted to determine the H-Ferritin receptor involved in this inflammation pathway. First, primary rat HSC (day 6, activated) were treated with AlexaFluor488 (AF488)-labeled recombinant human H-Ferritin. To identify H-Ferritin binding proteins, HSCs were incubated first with a membrane permeable (DMP, Dimethyl pimelimidate dihydrochloride) and then a membrane impermeable (BS³

(Bis[sulfosuccinimidyl] suberate)) cross-linker for 30 minutes each at 37°C. Cells were lysed, spun and membrane fraction proteins separated by electrophoresis, with gels scanned for AF488 fluorescence. Major fluorescently-labeled ferritin-crosslinked protein bands were excised, subjected to tryptic digest and identification by nanoHPLC MS/MS. None of the previously proposed H-Ferritin receptors were identified in our mass spectrometry analysis, suggesting that the H-Ferritin- receptor is specific for the inflammatory response. A receptor candidate identified in one band included the cell surface-bound adhesion molecule ICAM-1 (Figure 2A).

[0207] To validate these receptor candidates, ligand-receptor glycocapture technology (DualSystems Biotech) was performed using a cross-linking agent (TriCEPS™). TriCEPS contains three functional groups: an NHS ester for ligand conjugation {i.e., H-Ferritin), a hydrazide group to capture glycosylated receptors, and a biotin tag for analysis by LC-MS/MS. This approach revealed ICAM-1 as the only H-Ferritin receptor candidate within the dictated robust statistical parameters {i.e., area of volcano plot limited by the enrichment factor of > four-fold and a FDR-adjusted p- value of p < 0.01) (Figure 2B), also identified in the initial preliminary pull-down experiments followed by mass spectrometry analysis described above. Accordingly, these experiments reveal that ICAM-1 has a role as an H-Ferritin-receptor in the inflammation pathway.

Materials and Methods

[0208] Transfection of plasmids expressing ICAM-1. 3μg of pEGFP-C3 GFP or ICAM-1-GFP cDNAs (a kind gift from Prof. F. Sanchez-Madrid, CNIC, Spain) were transfected into HSC using LTX Lipofectamine according to the manufacturer's protocol . Experiments were performed next day.

[0209] FTH1 fluorescence labeling and Mass spectrometry. In preliminary studies we treated primary rat HSC (day 6, activated) with AlexaFluor488 (AF488)-labeled human H- Ferritin. To identify ferritin binding proteins, HSCs were incubated first with a membrane permeable (DMP, Dimethyl pimelimidate dihydrochloride) then a membrane impermeable (BS³ (Bis[sulfosuccinimidyl] suberate)) crosslinker for 30 min each at 37oC vs. 4oC. Cells were lysed, spun and membrane fraction proteins separated by electrophoresis, with gels scanned for AF488 fluorescence. Major fluorescently-labeled ferritin-crosslinked protein bands were excised, subjected to tryptic digest and identification by nanoHPLC MS/MS.

[0210] Ligand-receptor Glycocapture. H-Ferritin was conjugated with the TriCEPS™ reagent at 20°C. Cultured primary rat HSCs will be detached from tissue culture plates, oxidised with sodium metaperiodate prior to incubation with TriCEPS-coupled H-Ferritin (90 min, 4°C). Following HSC lysis and biotin-mediated affinity purification of ligand/receptor glycopeptides, peptides of interest were subjected to LC-MS/MS with ligand datasets statistically analyzed for candidates of interest.

EXAMPLE 3

MODULATING ICAM-1 ACTIVITY HAS SIGNIFICANT EFFECT ON INFLAMMATION

[0211] Strikingly, modulating ICAM-1 protein level by loss and gain of function experiments is clearly reflected in the expression and secretion of IL-Ιβ in response to H-Ferritin. Knock-down experiments of rat ICAM-1 in primary HSC, using a pool of three different siRNAs, resulted in a significantly decreased (~60%, p = 0.0021) H-Ferritin-induced expression of IL-Ιβ (Figure 2D) and NLRP3 (Figure 2E). Conversely, over-expression of human GFP-ICAM-1 in H- Ferritin-treated HSC led to a significant increase (~50%, p = 0.028) in IL-Ιβ expression (Figure 2C). Further to these data, ICAM-1 Knock-down resulted in >80% inhibition of H-Ferritin-induced IL-Ιβ protein secretion into conditioned medium in HSCs (Figure 2F). Finally, GFP-ICAM-1 augmentation of H-ferritin-induced ILi gene expression was inhibited by ~60% using the clathrin- coated pit inhibitor PitStop™ (Figure 2G), suggesting that receptor-mediated endocytosis via clathrin-coated pits is required for H-Ferritin to induced IL-Ιβ via ICAM-1. Materials and Methods

[0212] Methods as above. In addition, HSC were preincubated with 7.5μΜ PitStop (clathrin-coated pit endocytosis inhibitor) for 5min, prior to incubation with ΙΟηΜ H-ferritin for 2 hrs to assess the effect inhibiting clathrin-coated pit endocytosis on H-ferritin-induced IL-1 β gene expression (± GFP-ICAM overexpression).

EXAMPLE 4

ROLE OF ICAM-1 IN FTH-1-INDUCED INFLAMMASOME

[0213] To assess the role of FTHl in stimulating inflammasome activation primary rat HSC were treated with FTHl (10 nM, 24 hours), which stimulated the expression of NLRP3 protein. Changes in NLRPl protein were not observed. FTHl induced a marked increase in the expression of IL-Ιβ precursor protein, but of most interest caused a significant increase in the expression of the active (cleaved) form of IL-Ιβ protein (Figure 3).

[0214] The present investigations have revealed that ICAM-1 is the most promising candidate for the FTHl-receptor in HSCs. Further support for this has been derived via

manipulation of ICAM-1 protein levels by loss-of-function experiments (Figure 4). Knock-down of ICAM-1 (ICAM-1 kd) by ~75% in primary HSCs using a pool of three different siRNAs caused inhibition of FTHl-induced expression of NLRP3 protein levels. Figure 4 shows a marked FTH-1- induced increase in both the pro- and active (cleaved) forms of Caspase-1. ICAM-kd results in the complete loss of FTH-l-induced pro-Caspase-1 and marked decrease in active (cleaved) Capase-1. This demonstrates a clear role for ICAM-1 in the FTH-l-induced activation of the NLRP3 inflammasome in HSCs.

Materials and Methods

[0215] Western blotting. Primary HSCs were treated with FTHl (10 nM, 4 hours) to examine effects on components of the inflammasome. In separate experiments, primary HSC were transfected with either scrambled (SCR) or siRNA ICAM-1 (ICAM-kd) and treated with FTH-1 (10 nM, 24 hours). Whole primary rat HSC protein extracts were prepared by lysis of HSC cultures in RIPA buffer and lx sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris HCI pH6.8, 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% wt/vol bromophenol blue). Twenty micrograms of cell protein was subjected to SDS polyacrylamide gel electrophoresis and western blot using specific antibodies. Western blots were probed with anti-Caspase-1 (Adipogen, AG-20B- 0042-C100, 1 : 1000), anti-IL-Ιβ (Santa Cruz Biotechnology, sc-7884; 1 : 1000), anti-NLRP3 (Adipogen, AG-20B-0014; 1 : 1000), anti-NLRPl (Adipogen, AG-25B-0005, 1 : 1000), with anti-β- actin (Cell Signaling; 1 : 1000) as a loading control in TBST with 5% BSA powder. Blots were probed with an anti-rabbit/goat/mouse secondary at 1 : 5000-10000 in TBST 5% skim milk powder. HRP localization was detected by Western Lightning Plus ECL (Perkin Elmer) and visualized using X-ray film.

EXAMPLE 5

ROLE OF ICAM-1 DOMAINS IN FTH-1-INDUCED IL-Ιβ EXPRESSION

[0216] To determine the region of ICAM-1 activated by FTHl, gene expression constructs were made with targeted deletions of ICAM-1. Initially, cells were transfected with

ICAM-1 full length or mutants (Figure 5A). Compared to full length ICAM-1, the ICAM-1 domain 3 mutant caused a significant reduction in FTHl induced IL-Ιβ mRNA expression (Figure 5B). Subsequently, more specific mutants based on tertiary structure (Figure 5C) were transfected. Compared to full length ICAM-1, the ICAM-1 domain 4 (full) and the ICAM-1 domain 3+4 (full) caused a significant reduction in FTHl-induced IL-Ιβ mRNA expression (Figure 5D).

Materials and Methods

[0217] ICAM-1 mutant transfection. The full-length Human ICAM-1 cDNA sequence

(GenBank accession number: NM_000201) including the N-terminal signal peptide was cloned into the mammalian expression vector pcDNA3.1+ (Thermo Fisher Scientific). Initially the following mutants were made with the removed amino acid residues based on the nascent full-length polypeptide, as follows: Deletion Domain 1.1 (residues 1-103, corresponding to signal peptide and residues 1 to 76 of the mature polypeptide sequence), Deletion Domain 2.1 (129-201, corresponding to residues 102 to 174 of the mature polypeptide sequence), Deletion Domain 3.1 (240-305, corresponding to residues 213 to 278 of the mature polypeptide sequence), Deletion Domain 4.1 (335-388, corresponding to residues 308 to 361 of the mature polypeptide sequence), and Deletion Domain 5.1 (423-475, corresponding to residues 394 to 448 of the mature polypeptide sequence). More specific mutants based on tertiary structure were subsequently made with the removed amino acid residues based on the nascent full-length polypeptide noted in brackets: Deletion Domain 3.2 Full (209-310, corresponding to residues 183 to 283 of the mature polypeptide sequence), Deletion Domain 4.2 Full (309-393, corresponding to residues 282 to 366 of the mature polypeptide sequence), and Deletion Domains 3.2+4.2 Full (209-393, corresponding to residues 183 to 366 of the mature polypeptide sequence). Constructs were transfected into seven day post-isolation primary HSCs using Lipofectamine LTX (Thermo Fisher) and two days following transfection were treated with FTHl (10 nM) or control (water) for 2 hours.

[0218] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0219] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0220] 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. Those of skill in the art will therefore appreciate 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 such modifications and changes are intended to be included within the scope of the appended claims.

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Ruddell RG, Hoang-Le D, Barwood JM, eta/., Ferritin functions as a proinflammatory cytokine via iron-independent protein kinase C zeta/nuclear factor kappaB-regulated signaling in rat hepatic stellate cells. Hepato/ogy, 2009; 49 : 887-900.

Waalen J, Felitti VJ, Gelbart T, Beutler E. Screening for hemochromatosis by measuring ferritin levels: a more effective approach. Blood, 2008; 111 : 3373-6.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell.

2. A method of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, thereby inhibiting inflammasome assembly in the cell .

3. The method of claim 2, wherein the inflammasome comprises at least one {e.g., 1, 2, 3, etc.) component selected from caspase-1 (Casp-1), apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain (ASC), and NOD-like receptors) .

4. The method of claim 2 or claim 3, wherein the inflammasome comprises Casp-

1, ASC and any one or more of NLRP3, NLRP1 and NLRC4.

5. The method of any one of claims 1 to 4, wherein the cell is a hepatic stellate cell (HSC) a myofibroblast or a myofibroblast-like cell.

6. The method of any one of claims 1 to 5, wherein the ICAM-1 antagonist does not inhibit the interaction between the ICAM-1 polypeptide and a MAC-1 polypeptide.

7. The method of any one of claims 1 to 6, wherein the ICAM-1 antagonist does not inhibit the interaction between an ICAM-1 polypeptide and a LFA-1 polypeptide.

8. The method of any one of claims 1 to 7, wherein the method reduces expression of one or more inflammatory cytokine.

9. The method of claim 8, wherein the one or more inflammatory cytokine comprises interleukin-ΐβ (IL-Ιβ).

10. The method of any one of claims 1 to 8, wherein the ICAM-1 antagonist is an antigen-binding molecule that binds to a specific region of an ICAM-1 polypeptide.

11. The method of claim 10, wherein the antigen-binding molecule is a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody.

12. The method of claim 10 or claim 11, wherein the antigen-binding molecule is a monoclonal antibody.

13. The method of any one of claims 10 to 12, wherein the specific region of the ICAM-1 polypeptide is located fully or at least partially within domain 1 of the ICAM-1 polypeptide.

14. The method of any one of claims 10 to 12, wherein the specific region of the ICAM-1 polypeptide is located fully or at least partially within domain 2 of the ICAM-1 polypeptide.

15. The method of any one of claims 10 to 12, wherein the specific region of the

ICAM-1 polypeptide is located fully or at least partially within domain 3 of the ICAM-1 polypeptide.

16. The method of any one of claims 10 to 12, wherein the specific region of the ICAM-1 polypeptide is located fully or at least partially within domain 4 of the ICAM-1 polypeptide.

17. The method of any one of claims 10 to 12, wherein the specific region of the ICAM-1 polypeptide is located fully or at least partially within domain 5 of the ICAM-1 polypeptide.

18. The method of any one of claims 10 to 17, wherein the antigen-binding molecule does not compete with the R6.5 antibody (ATCC deposit no. HB-9580) for binding to the ICAM-1 polypeptide.

19. A method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 1 of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell.

20. A method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 2 of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell.

21. A method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 3 of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell.

22. A method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 4 of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell.

23. A method of inhibiting internalization of an H-Ferritin polypeptide into a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and the H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 5 of the ICAM-1 polypeptide, thereby inhibiting internalization of the H-Ferritin polypeptide into the cell.

24. A method of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 1 of ICAM-1, thereby inhibiting inflammasome assembly in the cell.

25. A method of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 2 of ICAM-1, thereby inhibiting inflammasome assembly in the cell.

26. A method of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 3 of ICAM-1, thereby inhibiting inflammasome assembly in the cell.

27. A method of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 4 of ICAM-1, thereby inhibiting inflammasome assembly in the cell.

28. A method of inhibiting inflammasome assembly in a cell that comprises a cell surface ICAM-1 polypeptide, the method comprising contacting the cell with an ICAM-1 antagonist that inhibits the interaction between the ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist binds to a region that is located fully or at least partially within domain 5 of ICAM-1, thereby inhibiting inflammasome assembly in the cell.

29. The method of any one of claims 24 to 28, wherein the inflammasome comprises at least one {e.g., 1, 2, 3, etc.) component selected from caspase-1 (Casp-1), apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain (ASC), and NOD-like receptors.

30. The method of any one of claims 24 to 29, wherein the inflammasome comprises Casp-1, ASC and any one of NLRP3, NLRP1 and NLRC4.

31. The method of any one of claims 19 to 30, wherein the cell is a hepatic stellate cell (HSC), myofibroblast or myofibroblast-like cell .

32. The method of any one of claims 19 to 31, wherein the ICAM-1 antagonist specifically inhibits the interaction between ICAM-1 and H-Ferritin and does not inhibit at least one other interaction of ICAM-1.

33. The method of claim 32, wherein the at least one other interaction of ICAM-1 includes the interaction between ICAM-1 and MAC-1.

34. The method of claim 32 and claim 33, wherein the at least one other interaction of ICAM-1 includes the interaction between ICAM-1 and LFA-1.

35. The method of any one of claims 19 to 34, wherein the method reduces expression of one or more inflammatory cytokines.

36. The method of claims 35, wherein the one or more inflammatory cytokines comprise interleukin-ΐβ (IL-Ιβ).

37. The method of any one of claims 19 to 36, wherein the ICAM-1 antagonist is an ICAM-1 antigen-binding molecule.

38. The method of claim 19 to 37, wherein the antigen-binding molecule is a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody.

39. The method of claim 37 or claim 38, wherein the antigen-binding molecule is a monoclonal antibody.

40. A method of producing an anti-inflammatory agent comprising : (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist in the presence of an H-Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

41. The method of claim 40, further comprising derivatizing the anti-inflammatory agent, and optionally formulating the derivatized anti-inflammatory agent with a

pharmaceutically acceptable carrier and/or diluent, to improve the efficacy of the agent or derivatized agent for inhibiting the inflammatory response.

42. The method of claim 40 or claim 41, wherein the candidate ICAM-1 antagonist is an antigen-binding molecule.

43. The method of claim 42, wherein the antigen-binding molecule is a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody.

44. The method of claim 42 or claim 43, wherein the antigen-binding molecule is a monoclonal antibody.

45. The method of claim 40 to claim 44, wherein the detecting of an inflammatory response includes the step of measuring the production of one or more inflammatory cytokine by the cell .

46. The method of claim 45, wherein the one or more inflammatory cytokine comprises IL-Ιβ.

47. The method of claim 40 to claim 44, wherein the detecting of an inflammatory response includes the step of measuring internalization of the H-Ferritin polypeptide into the cell, whereby the inflammatory response is detected as being inhibited if internalization of the H-Ferritin polypeptide is reduced.

48. The method of any one of claims 40 to 47, wherein the cell is an HSC, myofibroblast or myofibroblast cell .

49. A method of producing an anti-inflammatory agent comprising : (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 1 of the ICAM-1 polypeptide, in the presence of an H- Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

50. A method of producing an anti-inflammatory agent comprising : (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 2 of the ICAM-1 polypeptide, in the presence of an H- Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

51. A method of producing an anti-inflammatory agent comprising : (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 3 of the ICAM-1 polypeptide, in the presence of an H- Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

52. A method of producing an anti-inflammatory agent comprising : (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 4 of the ICAM-1 polypeptide, in the presence of an H- Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

53. A method of producing an anti-inflammatory agent comprising : (i) contacting a cell that comprises a cell surface ICAM-1 polypeptide with a candidate ICAM-1 antagonist that specifically binds to domain 5 of the ICAM-1 polypeptide, in the presence of an H- Ferritin polypeptide; and (ii) detecting inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of the candidate ICAM-1 antagonist, which indicates that the candidate ICAM-1 antagonist is an anti-inflammatory agent; and synthesizing or otherwise producing the anti-inflammatory agent.

54. The method of any one of claims 49 to 53, further comprising derivatizing the anti-inflammatory agent, and optionally formulating the derivatized anti-inflammatory agent with a pharmaceutically acceptable carrier and/or diluent, to improve the efficacy of the agent or derivatized agent for inhibiting the inflammatory response.

55. The method of any one of claims 49 to 54, wherein the anti-inflammatory response is detected by a method comprising measuring the production of one or more inflammatory cytokines by the cell .

56. The method of claim 55, wherein the one or more inflammatory cytokines comprises IL-Ιβ.

57. The method of any one of claims 49 to 54, wherein the detecting of an inflammatory response includes the step of measuring internalization of the H-Ferritin polypeptide into the cell, whereby the inflammatory response is detected as being inhibited if the internalization of the H-Ferritin polypeptide is reduced.

58. The method of any one of claims 49 to 57, wherein the cell is an HSC, myofibroblast or myofibroblast cell .

59. The method of claim 49 to 58, wherein the candidate ICAM-1 antagonist is an antigen-binding molecule.

60. The method of claim 59, wherein the antigen-binding molecule is a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody.

61. The method of claims 59 or claim 60, wherein the antigen-binding molecule is a monoclonal antibody.

62. A method of screening for an antagonist of binding between an ICAM-1 polypeptide and an H-Ferritin polypeptide, the method comprising incubating an ICAM-1 polypeptide and an H-Ferritin polypeptide in the presence of a candidate agent, and detecting whether the candidate agent inhibits binding of the ICAM-1 polypeptide with the H- Ferritin polypeptide, which indicates that the candidate agent is an antagonist of the binding between an ICAM-1 polypeptide and an H-Ferritin polypeptide.

63. The method of claim 62, wherein the ICAM-1 polypeptide is located on the surface of a cell .

64. The method of claim 63, wherein the cell is an HSC, myofibroblast or myofibroblast cell .

65. The method of any one of claim 63 or claim 64, wherein the detecting step comprises measuring an inhibition of an inflammatory response produced by the cell as compared to the inflammatory response produced by the cell in the absence of a candidate agent.

66. The method of claim 65, wherein the detection step comprises quantifying the production of one or more inflammatory cytokines.

67. The method of claim 66, wherein the one or more inflammatory cytokines include IL-Ιβ and/or IL-18.

68. A method of treating an inflammatory disease and/or condition in a subject, the method comprising administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, thereby treating the inflammatory disease and/or condition.

69. The method of claim 68, wherein the ICAM-1 antagonist does not inhibit the binding of the ICAM-1 polypeptide to a MAC-1 polypeptide.

70. The method of claim 68 and claim 69, wherein the at least one other interaction of ICAM-1 is the interaction between an ICAM-1 polypeptide to a LFA-1 polypeptide.

71. The method of any one of claims 68 to 70, wherein the method reduces the expression of one or more inflammatory cytokine.

72. The method of claim 71, wherein the one or more inflammatory cytokine comprises IL-Ιβ.

73. The method of any one of claims 68 to 72, wherein the ICAM-1 antagonist is an ICAM-1 antigen-binding molecule.

74. The method of claim 73, wherein the antigen-binding molecule is a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody.

75. The method of claims 73 or claim 74, wherein the antigen-binding molecule is a monoclonal antibody.

76. The method according to claim 75, wherein the antigen binding molecule binds region located fully or at least partially within domain 1 of the ICAM-1 polypeptide.

77. The method according to claim 75, wherein the antigen binding molecule binds region located fully or at least partially within domain 2 of the ICAM-1 polypeptide.

78. The method according to claim 75, wherein the antigen-binding molecule binds region located fully or at least partially within domain 3 of the ICAM-1 polypeptide.

79. The method according to claim 75, wherein the antigen-binding molecule binds region located fully or at least partially within domain 4 of the ICAM-1 polypeptide.

80. The method according to claim 75, wherein the epitope of ICAM-1 is located fully or at least partially within domain 5 of the ICAM-1 polypeptide.

81. The method of any one of claims 73 to 80, wherein the antigen-binding molecule does not compete with the R6.5 antibody (ATCC deposit no. HB-9580) for binding to the ICAM-1 polypeptide.

82. The method of any one of claims 68 to 81, wherein the inflammatory disease and/or condition is a chronic liver disease.

83. The method of claim 82, wherein the chronic liver disease is selected from fibrosis and cirrhosis.

84. A method of treating an inflammatory disease and/or condition in a subject, the method comprising administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 1 of the ICAM-1 polypeptide, thereby treating the inflammatory disease and/or condition in the subject.

85. A method of treating an inflammatory disease and/or condition in a subject, the method comprising administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 2 of the ICAM-1 polypeptide, thereby treating the inflammatory disease and/or condition in the subject.

86. A method of treating an inflammatory disease and/or condition in a subject, the method comprising administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 3 of the ICAM-1 polypeptide, thereby treating the inflammatory disease and/or condition in the subject.

87. A method of treating an inflammatory disease and/or condition in a subject, the method comprising administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 4 of the ICAM-1 polypeptide, thereby treating the inflammatory disease and/or condition in the subject.

88. A method of treating an inflammatory disease and/or condition in a subject, the method comprising administering to the subject an effective amount of an ICAM-1 antagonist that inhibits the interaction between an ICAM-1 polypeptide and an H-Ferritin polypeptide, wherein the ICAM-1 antagonist specifically binds to a region located fully or at least partially within domain 5 of the ICAM-1 polypeptide, thereby treating the inflammatory disease and/or condition in the subject.

89. The method of claim 88, wherein the ICAM-1 antagonist does not inhibit the binding of the ICAM-1 polypeptide to a MAC-1 polypeptide.

90. The method of claim 88 and claim 89, wherein the ICAM-1 antagonist does not inhibit the binding of the ICAM-1 polypeptide to a LFA-1 polypeptide.

91. The method of any one of claims 84 to 90, wherein the method reduces expression of one or more inflammatory cytokine.

92. The method of claim 91, wherein the one or more inflammatory cytokine comprises IL-Ιβ.

93. The method of claim 92, wherein the ICAM-1 antagonist is an antigen-binding molecule.

94. The method of claim 93, wherein the antigen-binding molecule is a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody.

95. The method of claims 93 or claim 94, wherein the antigen-binding molecule is a monoclonal antibody.

96. The method of any one of claims 84 to 95, wherein the inflammatory disease and/or condition is a chronic liver disease.

97. The method of claim 96, wherein the chronic liver disease is selected from fibrosis and cirrhosis.

98. Use of an anti-inflammatory agent produced by the method of any one of claims 40 to 61 in the manufacture of a medicament for the treatment of chronic liver disease.

99. The use of claim 98, wherein the chronic liver disease is selected from fibrosis and cirrhosis.

100. A purified or isolated antigen-binding molecule for use in reducing

inflammation in a subject, wherein the antigen-binding molecule binds to an ICAM-1 polypeptide and inhibits the binding of the ICAM-1 polypeptide to an H-Ferritin polypeptide, and wherein the antigen-binding molecule does not substantially inhibit the binding of the ICAM-1 polypeptide to one or both of a LFA1 polypeptide and a MAC-1 polypeptide.

101. The antigen-binding molecule of claim 100, wherein the antigen-binding molecule is selected from a monoclonal antibody, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')₂ fragment, diabody, intrabody, and a synthetic antibody.

102. The antigen-binding molecule of claim 101, wherein the antigen-binding molecule is a human, humanized or chimeric antibody.

103. The antigen-binding molecule of any one of claims 100 to 102, wherein the antigen-binding molecule does not substantially bind to an ICAM-2 polypeptide.

104. The antigen-binding molecule of any one of claims 100 to 103, wherein the antigen-binding molecule does not substantially bind to an ICAM-3 polypeptide.

105. The antigen-binding molecule of any one of claims 100 to 104, wherein the antigen-binding molecule does not substantially bind to an ICAM-5 polypeptide.

106. The antigen-binding molecule of any one of claims 100 to 105, wherein the antigen-binding molecule does not substantially bind to a VCAM-1 polypeptide.

107. The antigen-binding molecule of any one of claims 100 to 106, wherein the antigen-binding molecule inhibits the binding of the ICAM-1 polypeptide to a LFA1 polypeptide by no more than 20%.

108. The antigen-binding molecule of any one of claims 100 to 106, wherein the antigen-binding molecule inhibits the binding of the ICAM-1 polypeptide to the LFA1 polypeptide by no more than 10%.

109. The antigen-binding molecule of any one of claims 100 to 106, wherein the antigen-binding molecule inhibits the binding of the ICAM-1 polypeptide to the LFA1 polypeptide by no more than 5%.

110. The antigen-binding molecule of any one of claims 100 to 106 wherein the antigen-binding molecule inhibits the binding of the ICAM-1 polypeptide to the MACl polypeptide by no more than 20%.

111. The antigen-binding molecule of any one of claims 100 to 106, wherein the antigen-binding molecule inhibits the binding of the ICAM-1 polypeptide to the MACl polypeptide by no more than 10%.

112. The antigen-binding molecule of any one of claims 100 to 106, wherein the antigen-binding molecule inhibits the binding of the ICAM-1 polypeptide to the MACl polypeptide by no more than 5%.

113. An inflammatory agent produced by the method of any one of claims 40 to 61.