Il-32 Modulators

Abstract

The present invention relates to methods of screening for modulators of interleukin 32 (IL-32), to modulators of IL-32 and to their use. Proteinase 3 has been identified as an IL-32 binding protein capable of deavege thereof. Inhibition of PR 3 activity to process IL-32 or neutralization of IL-32 by-inactive PR3 or its fragments may reduce the consequences of IL-32 in immune regulated diseases (e.g. inflammatory diseases).

1 . A method or screening for a modulator of interleukin 32 (IL-32) activity based on the binding of IL-32 to proteinase 3 (PR-3), which comprises determining the binding of IL-32 to PR-3 in the presence of a candidate modulator, comparing the level of said binding to the level of binding of IL-32 to PR-3 in the absence of said candidate modulator, and selecting a modulator capable of inhibiting or enhancing said binding. 
     
     
         2 . The method according to  claim 1 , wherein said binding of IL-32 to PR-3 is measured by surface plasmon resonance. 
     
     
         3 . The method according to  claim 1 , wherein said modulator inhibits the binding of IL-32 to PR-3. 
     
     
         4 . The method according to  claim 3 , wherein said modulator is an inhibitor of IL-32 activity. 
     
     
         5 . The method according to  claim 4 , wherein said modulator is an inhibitor of the inflammatory activity of IL-32. 
     
     
         6 . The method according to  claim 1 , wherein said modulator enhances the binding of IL-32 to PR-3. 
     
     
         7 . The method according to  claim 6 , wherein said modulator is an enhancer of IL-32 activity. 
     
     
         8 . An inhibitor of IL-32 activity, selected by a method of screening for a modulator of interleukin 32 (IL-32) activity based on the binding of IL-32 to proteinase 3 (PR-3), which comprises determining the binding of IL-32 to PR-3 in the presence of a candidate modulator, comparing the level of said binding to the level of binding of IL-32 to PR-3 in the absence of said candidate modulator, and selecting a modulator capable of inhibiting or enhancing said binding, wherein said modulator is an inhibitor of IL-32 activity or wherein said modulator is an inhibitor of the inflammatory activity of IL-32. 
     
     
         9 . An enhancer of IL-32 activity, selected by a method of screening for a modulator of interleukin 32 (IL-32) activity based on the binding of IL-32 to proteinase 3 (PR-3), which comprises determining the binding of IL-32 to PR-3 in the presence of a candidate modulator, comparing the level of said binding to the level of binding of IL-32 to PR-3 in the absence of said candidate modulator, and selecting a modulator capable of inhibiting or enhancing said binding, wherein the modulator is an enhanced or IL-32 activity. 
     
     
         10 . A method of screening for an inhibitor of interleukin 32 (IL-32) activity based on the proteolytic activity of proteinase 3 (PR-3) on IL32, which comprises determining the proteolysis of IL-32 by PR-3 in the presence of a candidate inhibitor and selecting an inhibitor capable of inhibiting the appearance of an IL-32 fragment generated by the proteolityc activity of PR-3 or the disappearance of the intact IL-32. 
     
     
         11 . The method according to  claim 10 , wherein said inhibitor inhibits the inflammatory activity of IL-32. 
     
     
         12 . The method according to  claim 10  or  11 , wherein said IL-32 fragment is of about 16-kDa. 
     
     
         13 . The method according to  claim 10  or  11 , wherein said IL-32 fragment is of about 13 kDa. 
     
     
         14 . An inhibitor of IL-32 activity selected by the screening method according to  claim 10 . 
     
     
         15 . A method of screening for an inhibitor of interleukin 32 (IL-32) activity based on the enhancement of IL-32-mediated cytokine secretion by proteinase 3 (PR-3) in an IL-32 responsive cell, which comprises contacting IL-32 and PR-3 with a IL-32 responsive cell in the presence of a candidate inhibitor, determining the concentration of a cytokine in the culture medium of said cell, comparing to the concentration of said cytokine in the culture medium of said cell in the absence of said candidate inhibitor, and selecting for an inhibitor capable of inhibiting said cytokine secretion. 
     
     
         16 . The method according to  claim 15 , wherein said inhibitor inhibits the inflammatory activity of IL-32. 
     
     
         17 . The method according to  claim 15  or  16 , wherein the IL-32 responsive cell is a T cell or a macrophage cell. 
     
     
         18 . The method according to  claim 15 , wherein the cytokine is selected from the group consisting of TNF, IL-8 and MIP-2. 
     
     
         19 . An inhibitor of IL-32 activity selected by the method of screening according to  claim 15 . 
     
     
         20 . A method of screening for a modulator of the activity of interleukin 32 (IL-32) or of the activity of a fragment thereof, which comprises stimulating an IL-32-responsive cell with IL-32, or with a fragment thereof in the presence of a candidate modulator, determining the concentration of a cytokine secreted into the culture medium of said cell, comparing to the concentration of said cytokine secreted into the culture medium of said cell in the absence of said candidate modulator and selecting a modulator capable of inhibiting or enhancing secretion of said cytokine from said cell. 
     
     
         21 . The method according to  claim 20 , wherein the IL-32 fragment is a fragment of about 16 kDa generated by the proteolytic activity of PR-3. 
     
     
         22 . The method according to  claim 20 , wherein the IL-32 fragment is a fragment of about 13 kDa generated by the proteolytic activity of PR-3. 
     
     
         23 . The method according to  claim 20 , wherein the IL-32 responsive cell is a T cell or a macrophage cell. 
     
     
         24 . The method of screening according to  claim 20 , wherein said candidate modulator is selected from the group consisting of the inhibitors and enhancers of  claims 8 ,  9 ,  14 , and  19 . 
     
     
         25 . The method of screening according to  claim 20 , wherein the modulator is an inhibitor of the inflammatory activity of IL-32. 
     
     
         26 . A modulator of IL-32 activity selected by a method of screening according to  claim 20 . 
     
     
         27 . An inhibitor of IL-32 inflammatory activity selected by the method of screening according to  claim 25 . 
     
     
         28 - 41 . (canceled) 
     
     
         42 . A method for treating a disease which is caused or exacerbated by upregulated production and/or secretion of IL-32 or of a fragment thereof from cells that express it in a mammal, including a human, which comprises administering to such mammal in need an effective amount of a proteinase 3 (PR-3) inhibitor. 
     
     
         43 . The method according to  claim 42 , wherein the disease is caused or exacerbated by unregulated production and/or secretion of a fragment of IL-32. 
     
     
         44 . The method according to  claim 42  or  43 , wherein the IL-32 fragment is of about 16 kDa and is generated by the proteolytic activity of PR-3. 
     
     
         45 . The method according to  claim 42  or  43 , wherein the IL-32 fragment is of about 13 kDa and is generated by the proteolytic activity of PR-3. 
     
     
         46 . The method according to  claim 42 , wherein the cells that express IL-32 or a fragment thereof are epithelial cells. 
     
     
         47 . The method according to  claim 42 , wherein the PR-3 inhibitor is selected from the group consisting of isocoumarin, dichloroisocoumarin, suramin, hexasulfonated naphtylurea, peptidomimetic agents based on 1,2,5-thiadiazolidin-3-one 1,1-dioxide backbone and their sulfone derivatives, proteinase inhibitor, elafin, antileukoprotease eglin C, MNEI (monocyte/neutrophile elastase inhibitor), the bioengineered serpin LEX032, and a neutralizing anti-PR-3 antibody. 
     
     
         48 . A method for treating a disease which is caused or exacerbated by unregulated production and/or secretion of IL-32 or of a fragment thereof from cells that express it in a mammal, including a human, which comprises administering to such mammal in need an effective amount of a modulator according to any one of  claims 8 ,  9 ,  14 ,  19  and  26 . 
     
     
         49 . The method according to  claim 48 , wherein the disease is caused or exacerbated by upregulated production and/or secretion of a fragment of IL-32. 
     
     
         50 . The method according to  claim 49 , wherein the cells that express IL-32 are epithelial cells. 
     
     
         51 . The method according to  claim 48 , wherein the IL-32 fragment is of about 16 kDa and is generated by the proteolytic activity of PR-3. 
     
     
         52 . The method according to  claim 48 , wherein the IL-32 fragment is of about 13 kDa and is generated by the proteolytic activity of PR-3. 
     
     
         53 . A polypeptide fragment of IL-32 obtained by the proteolysis of IL-32 by proteinase 3 (PR-3). 
     
     
         54 . A polypeptide fragment according to  claim 53 , wherein the IL-32 fragment consists of about 16 kDa obtained by the proteolysis of IL-32 by proteinase 3 (PR-3) or, a mutein, fusion protein, functional derivative, a circularly permuted derivative, or active fraction thereof. 
     
     
         55 . A polypeptide fragment according to  claim 53 , wherein the IL-32 fragment consists of about 13 kDa obtained by the proteolysis of IL-32 by proteinase 3 (PR-3) or, a mutein, fusion protein, functional derivative, a circularly permuted derivative, or active fraction thereof. 
     
     
         56 . A pharmaceutical composition comprising a polypeptide fragment according to  claim 53  and a pharmaceutically acceptable carrier. 
     
     
         57 . A polypeptide fragment of IL-32 of SEQ ID NO: 1 or, a mutein, fusion protein, functional derivative, a circularly permuted derivative, or active fraction thereof. 
     
     
         58 . A polypeptide fragment of IL-32 of SEQ ID NO: 2 or, a mutein, fusion protein, functional derivative, a circularly permuted derivative, or active fraction thereof. 
     
     
         59 . A pharmaceutical composition comprising polypeptide fragment of IL-32 of SEQ ID NO: 1 or, a mutein, fusion protein, functional derivative, a circularly permuted derivative, or active fraction thereof and a pharmaceutically acceptable carrier. 
     
     
         60 . A pharmaceutical composition comprising polypeptide fragment of IL-32 of SEQ ID NO: 2 or, a mutein, fusion protein, functional derivative, a circularly permuted derivative, or active fraction thereof and a pharmaceutically acceptable carrier. 
     
     
         61 . A modulator of IL-32 activity comprising a fragment of PR-3 capable of binding IL-32, but not of cleaving 11-32.