Removal promoters and inhibitor for apoptosis cells in vivo

Abstract

The present invention is to provide a removal promoter for apoptotic cells which is capable of immediately removing apoptotic cells in vivo by macrophages, or a removal inhibitor which inhibits the removal of apoptotic cells in vivo by macrophages. A removal promoter for apoptotic cells in vivo containing the milk fat globule-EGF factor 8-L (MFG-E8-L), MFG-E8-L mutant having removal promotion action for apoptotic cells in vivo by macrophages, or preferably a recombinant human or mouse MFG-E8-L, or a recombinant human or mouse MFG-E8-L mutant as an active ingredient is prepared. Such removal promoters specifically bind to apoptotic cells and promote the phagocytosis of apoptotic cells by macrophages by recognizing aminophospholipids such as phosphatidylserine exposed on apoptotic cell surface. On the other hand, a point mutation (D89E) MFG-E8-L mutant is used as a removal inhibitor.

1 . A removal promoter for apoptotic cells in vivo by macrophages which contains MFG-E8-L as an active ingredient.  
     
     
         2 . A removal promoter for apoptotic cells in vivo by macrophages which is comprised of amino acid sequence wherein one or more amino acids are deleted, substituted or added in the amino acid sequence comprising MFG-E8-L, and which contains MFG-E8-L mutant having removal promotion action for apoptotic cells in vivo by macrophages as an active ingredient.  
     
     
         3 . The removal promoter for apoptotic cells in vivo by macrophages according to  claim 1  wherein the MFG-E8-L or the MFG-E8-L mutant which has removal action for apoptotic cells is a recombinant MFG-E8-L or a recombinant MFG-E8-L mutant.  
     
     
         4 . The removal promoter for apoptotic cells in vivo by macrophages according to  claim 3  wherein the recombinant MFG-E8-L or the recombinant MFG-E8-L mutant is a recombinant human or mouse MFG-E8-L, or a recombinant human or mouse MFG-E8-L mutant.  
     
     
         5 . The removal promoter for apoptotic cells in vivo by macrophages according to  claim 3  wherein the recombinant MFG-E8-L or the recombinant MFG-E8-L mutant is a translation product in human cells.  
     
     
         6 . The removal promoter for apoptotic cells in vivo by macrophages according to  claim 3  wherein the recombinant MFG-E8-L or the recombinant MFG-E8-L mutant contains an EGF-2 domain having RGD motif, a proline/threonine-rich domain, and two factor VIII-homologous domains (C1 and C2).  
     
     
         7 . The removal promoter for apoptotic cells in vivo by macrophages according to  claim 1  wherein the MFG-E8-L or the MFG-E8-L mutant is enveloped or embedded in liposome.  
     
     
         8 . The removal promoter for apoptotic cells in vivo by macrophages which contains a recombinant vector including DNA encoding the MFG-E8-L or MFG-E8-L mutant according to  claim 1  as an active ingredient.  
     
     
         9 . The removal promoter for apoptotic cells in vivo by macrophages which contains a host cell comprising the expression system which can express the MFG-E8-L or the MFG-E8-L mutant according to  claim 1  as an active ingredient.  
     
     
         10 . The removal promoter for apoptotic cells in vivo by macrophages which contains an antibody against the MFG-E8-L mutant according to  claim 1  as an active ingredient.  
     
     
         11 . The removal promoter for apoptotic cells in vivo by macrophages according to  claim 10 .  
     
     
         12 . A removal method for apoptotic cells in vivo by macrophages wherein the removal promoter for apoptotic cells in vivo according to  claim 1  is used.  
     
     
         13 . A therapeutic agent for diseases resulting from incomplete removal of apoptotic cells in vivo by macrophages which contains the removal promoter for apoptotic cells in vivo by macrophages according to  claim 1 .  
     
     
         14 . An enhancer for biodefense mechanism which contains the removal promoter for apoptotic cells in vivo according to  claim 1 .  
     
     
         15 . A therapeutic method for diseases resulting from incomplete removal of apoptotic cells in vivo by macrophages wherein the therapeutic agent according to  claim 13  is used.  
     
     
         16 . A removal inhibitor for apoptotic cells in vivo by macrophages comprised of amino acid sequence wherein one or more amino acids are deleted, substituted or added in the amino acid sequence comprising MFG-E8-L, and which contains a MFG-E8-L mutant having removal inhibition action for apoptotic cells in vivo by macrophages as an active ingredient.  
     
     
         17 . The removal inhibitor for apoptotic cells in vivo by macrophages according to  claim 16  wherein the MFG-E8-L mutant having removal inhibition action for apoptotic cells is a recombinant MFG-E8-L mutant.  
     
     
         18 . The removal inhibitor for apoptotic cells in vivo by macrophages according to  claim 17  wherein the recombinant MFG-E8-L mutant is a recombinant human MFG-E8-L mutant or a recombinant mouse MFG-E8-L mutant.  
     
     
         19 . The removal inhibitor for apoptotic cells in vivo by macrophages according to  claim 17  wherein the recombinant MFG-E8-L mutant is a translation product in human cells.  
     
     
         20 . The removal inhibitor for apoptotic cells in vivo by macrophages according to  claim 17  wherein the recombinant MFG-E8-L mutant is a MFG-E8-L mutant which contains a proline/threonine-rich domain and two factor VIII-homologous domains (C1 and C2), and which has a point mutation in RGD motif.  
     
     
         21 . The removal inhibitor for apoptotic cells in vivo by macrophages according to  claim 20  wherein the MFG-E8-L mutant which has a point mutation is D89E mutant.  
     
     
         22 . The removal inhibitor for apoptotic cells in vivo by macrophages according to  claim 16  wherein the MFG-E8-L mutant is enveloped or embedded in liposome.  
     
     
         23 . The removal inhibitor for apoptotic cells in vivo by macrophages which contains a recombinant vector including DNA encoding the MFG-E8-L mutant according to  claim 16  as an active ingredient.  
     
     
         24 . The removal inhibitor for apoptotic cells in vivo by macrophages which contains a host cell comprising an expression system which can express the MFG-E8-L mutant according to  claim 16  as an active ingredient.  
     
     
         25 . A removal inhibition method for apoptotic cells in vivo by macrophages wherein the removal inhibitor for apoptotic cells in vivo according to  claim 16  is used.  
     
     
         26 . A therapeutic agent for diseases resulting from the incomplete removal inhibition of apoptotic cells in vivo by macrophages which contains the removal inhibitor for apoptotic cells in vivo according to  claim 16 .  
     
     
         27 . A therapeutic method for diseases resulting from the incomplete removal inhibition of apoptotic cells in vivo by macrophages wherein the therapeutic agent according to  claim 26  is used.  
     
     
         28 . A detection agent for apoptotic cells in vivo which contains a labeled MFG-E8-L or MFG-E8-L mutant having removal promotion action for apoptotic cells in vivo by macrophages, or an antibody against them, or a labeled MFG-E8-L mutant having removal inhibition action for apoptotic cells in vivo by macrophages as an active ingredient.  
     
     
         29 . A detection method for apoptotic cells in vivo wherein a detection agent for apoptotic cells in vivo which contains a labeled MFG-E8-L or MFG-E8-L mutant having removal promotion action for apoptotic cells in vivo by macrophages, or an antibody against them, or a labeled MFG-E8-L mutant having removal inhibition action for apoptotic cells in vivo by macrophages as an active ingredient is used.  
     
     
         30 . A screening method for a removal promotion inducing substance or a removal promotion suppressive substance for apoptotic cells in vivo by macrophages wherein MFG-E8-L or a MFG-E8-L mutant having removal promotion action for apoptotic cells in vivo by macrophages, or an antibody against them is contacted with a test substance, to evaluate the extent of removal of apoptotic cells in vivo.  
     
     
         31 . A screening method for a removal inhibition inducing substance or a removal inhibition suppressive substance for apoptotic cells in vivo by macrophages wherein a MFG-E8-L mutant which has removal inhibition action for apoptotic cells in vivo by macrophages is contacted with a test substance, to evaluate the extent of removal inhibition of apoptotic cells in vivo.