Method of extensive culture of antigen-specific cytotoxic T cells

Abstract

The present invention provides methods for inducing, maintaining and expanding CTL (cytotoxic T cell) having an antigen-specific cytotoxic activity at a high level, which is useful in the adoptive immunotherapy, by using as an effective ingredient at least one compound selected from the group consisting of acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides, and salts thereof. The above-mentioned compounds include fucoidans, heparins, alginic acid, chondroitin sulfate A, chondroitin sulfate B, pectic acid, hyaluronic acid, degradation products of fucoidans, sulfated glucose, sulfated fucose and salts thereof.

1 . A method for inducing a cytotoxic T cell having an antigen-specific cytotoxic activity from a precursor cell capable of differentiating to a cytotoxic T cell, which comprises:
 incubating a precursor cell capable of differentiating to cytotoxic T cell with an antigen presenting cell in the presence of at least one compound selected from the group consisting of acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides, and salts thereof.   
     
     
         2 . The method according to  claim 1 , wherein the acidic polysaccharide is at least one compound selected from the group consisting of fucoidan, F-fucoidan, U-fucoidan, Low Molecular Weight heparin, alginic acid, chondroitin sulfate A, chondroitin sulfate B, pectic acid and salts thereof. 
     
     
         3 . The method according to  claim 1 , wherein the at least one compound is selected from the group consisting of: sulfated glucose, sulfated fucose, Formula IV and salts thereof, wherein Formula IV has the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         4 . A method for maintaining cytotoxic T cell having an antigen-specific cytotoxic activity, wherein the method comprises:
 (1) incubating a precursor cell capable of differentiating to cytotoxic T cell with an antigen presenting cell in the presence of at least one compound selected from the group consisting of acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides and salts thereof, to induce a cytotoxic T cell having an antigen-specific cytotoxic activity, and   (2) continuously culturing the induced cytotoxic T cell obtained from step (1) in the presence of at least one compound selected from the group consisting of acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides, and salts thereof.   
     
     
         5 . The method according to  claim 4 , wherein the acidic polysaccharide is at least one compound selected from the group consisting of fucoidan, F-fucoidan, U-fucoidan, Low Molecular weight heparin, alginic acid, chondroitin sulfate A, chondroitin sulfate B, pectic acid and salts thereof. 
     
     
         6 . The method according to  claim 4 , wherein the at least one compound is selected from the group consisting of: sulfated glucose, sulfated fucose, Formula IV and salts thereof, wherein Formula IV has the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         7 . A method for expanding a cytotoxic T cell having an antigen-specific cytotoxic activity, which comprises:
 (1) incubating a precursor cell capable of differentiating to cytotoxic T cell with an antigen presenting cell in the presence of at least one compound selected from the group consisting of acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides and salts thereof, to induce a cytotoxic T cell having an antigen-specific cytotoxic activity, and   (2) incubating the induced cytotoxic T cell obtained by step (1) in the presence of at least one compound selected from the group consisting of acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides and salts thereof.   
     
     
         8 . The method according to  claim 7 , wherein the cytotoxic T cell is incubated further in the presence of anti-CD3 antibody in said step. 
     
     
         9 . The method according to  claim 7 , wherein the at least one compound is selected from the group consisting of: sulfated glucose, sulfated fucose, Formula IV and salts thereof, wherein Formula IV has the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         10 . The method according to  claim 7 , wherein the cytotoxic T cell is incubated together with a feeder cell in said step. 
     
     
         11 . The method according to  claim 7 , wherein the feeder cell is a non-virus-infected cell. 
     
     
         12 . The method according to  claim 7 , wherein the acidic polysaccharide is at least one compound selected from the group consisting of fucoidan, F-fucoidan, U-coidan, Low Molecular weight heparin, alginic acid, chondroitin sulfate A, chondroitin sulfate B, pectic acid and salts thereof. 
     
     
         13 . A method for collecting a cytotoxic T cell, which comprises:
 selecting a cell population rich in cytotoxic T cells having an antigen-specific cytotoxic activity from a culture containing the cytotoxic T cell obtained by the method of any one of  claims 1 ,  4  and  7 ; and   collecting said cell population.   
     
     
         14 . A cytotoxic T cell having antigen-specific cytotoxic activity prepared by the method of  claim 1 . 
     
     
         15 . A therapeutic agent, characterized in that the therapeutic agent comprises the cytotoxic T cell of  claim 14  as an effective ingredient. 
     
     
         16 . A method for expanding a cytotoxic T cell having an antigen-specific cytotoxic activity, which comprises:
 incubating the cytotoxic T cell having an antigen-specific cytotoxic activity in the presence of at least one compound selected from the group consisting of: acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides and salts thereof.   
     
     
         17 . The method according to  claim 16 , which further comprises incubating the cytotoxic T cell in the presence of anti-CD3 antibody. 
     
     
         18 . The method according to  claim 16 , which further comprises incubating the cytotoxic T cell together with a feeder cell. 
     
     
         19 . The method according to  claim 18 , wherein the feeder cell is a non-virus-infected cell. 
     
     
         20 . A method for collecting a cytotoxic T cell, which comprises:
 selecting a cell population rich in cytotoxic T cells having an antigen-specific cytotoxic activity from a culture containing the cytotoxic T cell obtained by the method of  claim 16 .   
     
     
         21 . A cytotoxic T cell having antigen-specific cytotoxic activity prepared by the method of  claim 16 . 
     
     
         22 . A therapeutic composition, comprising the cytotoxic T cell of  claim 21  as an effective ingredient and a pharmaceutically acceptable carrier therefore. 
     
     
         23 . The method according to  claim 16 , wherein the at least one compound is selected from the group consisting of: sulfated glucose, sulfated fucose, Formula IV and salts thereof, wherein Formula IV has the following structure: