Method for improving affinity of antibody for antigen and use thereof

Abstract

The present invention relates to a method for improving affinity of an antibody for an antigen. The present invention relates to a modified antibody with improved affinity for an antigen as compared to an unmodified antibody. The present invention relates to a method for producing an antibody with improved affinity for an antigen as compared to an unmodified antibody. The present invention relates to a method for analyzing an amino acid sequence of an antibody. The present invention relates to a method for specifying candidates for an antibody modification site.

1 . A method for improving affinity of an antibody for an antigen as compared to an unmodified antibody by, in an amino acid sequence of a framework region of a light chain of the antibody, changing at least 3 of amino acid residues present at positions where total value of amino acid frequencies of arginine, serine, threonine, valine, aspartic acid and glutamic acid is 35% or more and having a ratio of solvent-exposed surface area of 20% or more to charged amino acid residues. 
     
     
         2 . The method according to  claim 1 , wherein
 the amino acid residues present at positions where the total value of amino acid frequencies is 35% or more are specified by:   obtaining amino acid sequences of light chains of a plurality of reference antibodies;   aligning the amino acid sequences of light chains of a plurality of reference antibodies to calculate total value of amino acid frequencies of arginine, serine, threonine, valine, aspartic acid and glutamic acid at each position of the amino acid sequences of framework regions;   specifying positions where the obtained total value of amino acid frequencies is 35% or more; and   specifying amino acid residues present at positions corresponding to the positions specified from the plurality of reference antibodies, in a framework region of a light chain of the unmodified antibody.   
     
     
         3 . The method according to  claim 1  or  2 , wherein the amino acid residues having a ratio of solvent-exposed surface area of 20% or more are specified based on an amino acid sequence of a light chain of the unmodified antibody. 
     
     
         4 . The method according to any one of  claims 1  to  3 , wherein
 the amino acid residues having a ratio of solvent-exposed surface area of 20% or more are specified by: 
 obtaining three-dimensional structure data of a light chain of the unmodified antibody by using an amino acid sequence of a light chain of the unmodified antibody; 
 obtaining a ratio of solvent-exposed surface area of each amino acid residue of the framework region based on the three-dimensional structure data; and 
 specifying amino acid residues having an obtained ratio of solvent-exposed surface area of 20% or more. 
 
     
     
         5 . A modified antibody with improved affinity for an antigen as compared to an unmodified antibody, wherein,
 in an amino acid sequence of a framework region of a light chain of the unmodified antibody, at least 3 of amino acid residues present at positions where total value of amino acid frequencies of arginine, serine, threonine, valine, aspartic acid and glutamic acid is 35% or more and having a ratio of solvent-exposed surface area of 20% or more are changed to charged amino acid residues.   
     
     
         6 . A method for producing an antibody with improved affinity for an antigen as compared to an unmodified antibody, comprising:
 in an amino acid sequence of a framework region of a light chain of the antibody, changing at least 3 of amino acid residues present at positions where total value of amino acid frequencies of arginine, serine, threonine, valine, aspartic acid and glutamic acid is 35% or more and having a ratio of solvent-exposed surface area of 20% or more to charged amino acid residues; and   recovering the antibody obtained in the changing.   
     
     
         7 . A method for analyzing an amino acid sequence of an antibody, comprising:
 obtaining a total value of amino acid frequencies of arginine, serine, threonine, valine, aspartic acid and glutamic acid at each position in an amino acid sequence of a framework region of a light chain of an antibody;   obtaining a ratio of solvent-exposed surface area of each amino acid residue in the framework region of a light chain of an antibody; and   outputting the obtained total value of amino acid frequencies at each position and the obtained ratio of solvent-exposed surface area of each amino acid residue.   
     
     
         8 . The method according to  claim 7 , wherein
 the total value of amino acid frequencies of the antibody is obtained by:   obtaining amino acid sequences of light chains of a plurality of reference antibodies;   aligning the amino acid sequences of light chains of a plurality of reference antibodies to calculate a total value of amino acid frequencies of arginine, serine, threonine, valine, aspartic acid and glutamic acid at each position of the amino acid sequences of framework regions; and   obtaining the calculated total value of amino acid frequencies as a total value of amino acid frequencies of the plurality of reference antibodies at each position of the amino acid sequence of a framework region of an antibody corresponding to the each position.   
     
     
         9 . The method according to  claim 8 , wherein the amino acid sequences of light chains of a plurality of reference antibodies contain an amino acid sequence downloaded from an external database and/or an amino acid sequence input by a user. 
     
     
         10 . The method according to any one of  claims 7  to  9 , wherein, in the obtaining a ratio of solvent-exposed surface area of an antibody, the ratio of solvent-exposed surface area of an antibody is obtained based on the amino acid sequence of a framework region of a light chain of an antibody. 
     
     
         11 . The method according to any one of  claims 7  to  10 , further comprising obtaining three-dimensional structure data of a light chain of the antibody by using the amino acid sequence of a light chain of the antibody,
 wherein, in the obtaining a ratio of solvent-exposed surface area of an antibody, the ratio of solvent-exposed surface area of each amino acid residue of the framework region is obtained based on the three-dimensional structure data. 
 
     
     
         12 . The method according to  claim 11 , wherein
 the three-dimensional structure data of a light chain of the antibody contains at least one selected from following data:   three-dimensional structure data retrieved from an external database using the amino acid sequence of a light chain of the antibody and downloaded from the external database;   three-dimensional structure data created based on information necessary for creating the three-dimensional structure data of a light chain of the antibody downloaded from the external database; and   three-dimensional structure data input by the user.   
     
     
         13 . A method for specifying candidates for an antibody modification site, comprising:
 obtaining a total value of amino acid frequencies of arginine, serine, threonine, valine, aspartic acid and glutamic acid at each position in an amino acid sequence of a framework region of a light chain of an antibody;   obtaining a ratio of solvent-exposed surface area of each amino acid residue in the framework region of a light chain of an antibody; and   specifying the amino acid residues present at positions where the total value of amino acid frequencies is 35% or more and having a ratio of solvent-exposed surface area of 20% or more as candidates for amino acid residues to be modified in order to improve affinity of the antibody for an antigen.   
     
     
         14 . The method according to  claim 13 , further comprising outputting the specified candidates for amino acid residues. 
     
     
         15 . The method according to  claim 13  or  14 , wherein the amino acid sequence of a framework region of a light chain of an antibody is obtained from the amino acid sequence of a light chain of the antibody input by a user. 
     
     
         16 . The method according to  claim 15 , further comprising creating a modified amino acid sequence when at least 3 of the candidates for amino acid residues are changed to charged amino acids, in the amino acid sequence of a light chain of the antibody input by the user. 
     
     
         17 . The method according to  claim 16 , further comprising outputting the created modified amino acid sequence. 
     
     
         18 . The method according to any one of  claims 13  to  17 , wherein
 the amino acid residues present at positions where the total value of amino acid frequencies is 35% or more are specified by: 
 obtaining amino acid sequences of light chains of a plurality of reference antibodies; 
 aligning the amino acid sequences of light chains of a plurality of reference antibodies to calculate total value of amino acid frequencies of arginine, serine, threonine, valine, aspartic acid and glutamic acid at each position of the amino acid sequences of framework regions; 
 specifying positions where the obtained total value of amino acid frequencies is 35% or more; and 
 specifying amino acid residues present at positions corresponding to the positions specified from the plurality of reference antibodies, in the framework region of a light chain of the antibody. 
 
     
     
         19 . The method according to  claim 18 , wherein the amino acid sequences of light chains of a plurality of reference antibodies contain an amino acid sequence downloaded from an external database and/or an amino acid sequence input by a user. 
     
     
         20 . The method according to any one of  claims 13  to  19 , wherein
 the amino acid residues having a ratio of solvent-exposed surface area of 20% or more are specified by: 
 obtaining three-dimensional structure data of a light chain of the antibody by using the amino acid sequence of a light chain of the antibody; 
 obtaining a ratio of solvent-exposed surface area of each amino acid residue of the framework region based on the three-dimensional structure data; and 
 specifying amino acid residues having an obtained ratio of solvent-exposed surface area of 20% or more. 
 
     
     
         21 . The method according to  claim 20 , wherein
 the three-dimensional structure data of a light chain of the antibody contains at least one selected from following data:   three-dimensional structure data retrieved from an external database using the amino acid sequence of a light chain of the antibody and downloaded from the external database;   three-dimensional structure data created based on information necessary for creating the three-dimensional structure data of a light chain of the antibody downloaded from the external database; and   three-dimensional structure data input by the user.   
     
     
         22 . The method according to any one of  claims 13  to  21 , further comprising calculating electrical characteristic of complementarity determining region (CDR) from the amino acid sequence of a light chain of the antibody,
 wherein the electrical characteristic is determined by following formula (III):
     Z =[Number of basic amino acid residues in amino acid sequence of CDR]−[Number of acidic amino acid residues in amino acid sequence of CDR]  (III)
 
 
 wherein when Z is −1, 0 or 1, the electrical characteristic of CDR is neutral, 
 when Z is 2 or more, the electrical characteristic of CDR is positively charged, and 
 when Z is −2 or less, the electrical characteristic of CDR is negatively charged. 
 
     
     
         23 . The method according to  claim 22 , further comprising outputting the calculated electrical characteristic. 
     
     
         24 . The method according to  claim 22  or  23 , wherein, when the calculated electrical characteristic of CDR is neutral, a modified amino acid sequence when at least 3 of the candidates for amino acid residues are changed to basic amino acid residues is created. 
     
     
         25 . A modified antibody with improved affinity for an antigen as compared to an unmodified antibody, wherein
 positions of modified amino acid residues are positions of at least 3 amino acid residues of the candidates for amino acid residues specified by the method according to any one of  claims 13  to  24 , and   the modification is modification to a charged amino acid residue.   
     
     
         26 . A method for producing an antibody with improved affinity for an antigen as compared to an unmodified antibody, comprising:
 changing at least 3 of the candidates for amino acid residues specified by the method according to any one of  claims 13  to  24  to charged amino acid residues; and   recovering the antibody obtained in the changing.   
     
     
         27 . A method for improving affinity of an antibody for an antigen, comprising:
 in an unmodified antibody, improving affinity for an antigen as compared to the unmodified antibody, by changing at least 3 amino acid residues selected from a group consisting of 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 63rd, 65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th and 81st amino acid residues of a light chain defined by Kabat method to charged amino acid residues,   wherein the 3 amino acid residues contain at least one selected from a group consisting of 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 74th, 76th, 77th, 79th and 81st amino acid residues of the light chain.   
     
     
         28 . The method according to  claim 27 , wherein the 3 amino acid residues are selected from a group consisting of 3rd, 5th, 9th, 17th, 18th, 20th and 22nd amino acid residues of the light chain. 
     
     
         29 . A modified antibody with improved affinity for an antigen as compared to an unmodified antibody, wherein
 at least 3 amino acid residues selected from a group consisting of 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 63rd, 65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th and 81st amino acid residues of a light chain defined by Kabat method in the unmodified antibody are changed to charged amino acid residues, and the 3 amino acid residues contain at least one selected from a group consisting of 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 74th, 76th, 77th, 79th and 81st amino acid residues of the light chain.   
     
     
         30 . A method for producing an antibody with improved affinity for an antigen as compared to an unmodified antibody, comprising:
 in an unmodified antibody, changing at least 3 amino acid residues selected from a group consisting of 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 63rd, 65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th and 81st amino acid residues of a light chain defined by Kabat method to charged amino acid residues; and   recovering the antibody obtained in the changing,   wherein the 3 amino acid residues contain at least one selected from a group consisting of 3rd, 5th, 9th, 17th, 18th, 20th, 22nd, 60th, 74th, 76th, 77th, 79th and 81st amino acid residues of the light chain.   
     
     
         31 . The method according to any one of  claims 1  to  4 ,  6 ,  16 ,  26 ,  27  and  30 , or the antibody according to any one of  claims 5 ,  25  and  29 , wherein the charged amino acid residue is a basic amino acid residue. 
     
     
         32 . The method according to any one of  claims 1  to  4 ,  6 ,  16 ,  24 ,  26 ,  27 ,  30  and  31 , or the antibody according to any one of  claims 5 ,  25 ,  29  and  31 , wherein the framework region is framework region 1 or framework region 3.