Antigen-binding molecule capable of binding to two or more antigen molecules repeatedly

Abstract

The present inventors discovered that antibodies having weaker antigen-binding activity at the early endosomal pH in comparison with that at the pH of plasma are capable of binding to multiple antigen molecules with a single antibody molecule, have long half-lives in plasma, and have improved durations of time in which they can bind to antigen.

1 .- 27 . (canceled) 
     
     
         28 . A method of screening for an antibody, which comprises the steps of:
 (a) determining the antigen-binding activity of an antibody at pH 6.7 to pH 10.0;   (b) determining the antigen-binding activity of the antibody at pH 4.0 to pH 6.5; and   (c) selecting an antibody whose antigen-binding activity at pH 6.7 to pH 10.0 is greater than the antigen-binding activity at pH 4.0 to pH 6.5, wherein the antibody is used as a pharmaceutical composition and is any one of the following (i) to (vi):   (i) an antibody that is superior in retention in plasma;   (ii) an antibody that is capable of binding to an antigen two or more times when measured in a nonhuman animal comprising a cell expressing FcRn;   (iii) an antibody that is capable of binding to more antigens as compared to the number of its antigen-binding sites when measured in a nonhuman animal comprising a cell expressing FcRn;   (iv) an antibody that dissociates an extracellularly-bound antigen within a cell;   (v) an antibody that is bound to an antigen and internalized into a cell, and released to the outside of the cell in an antigen-free form; or   (vi) an antibody that has increased ability to eliminate an antigen in plasma.   
     
     
         29 . The screening method of  claim 28 , which comprises the step of selecting an antibody whose antigen-binding activity at pH 6.7 to pH 10.0 is twice or higher that of the antigen-binding activity at pH 4.0 to pH 6.5. 
     
     
         30 . A method of screening for an antibody, which comprises the steps of:
 (a) binding an antibody to an antigen under a condition of pH 6.7 to pH 10.0;   (b) placing the antibody bound to the antigen of (a) under a condition of pH 4.0 to pH 6.5; and   (c) obtaining an antibody that has dissociated under the condition of pH 4.0 to pH 6.5, wherein the antibody is used as a pharmaceutical composition and is any one of the following (i) to (vi):   (i) an antibody that is superior in retention in plasma;   (ii) an antibody that is capable of binding to an antigen two or more times when measured in a nonhuman animal comprising a cell expressing FcRn;   (iii) an antibody that is capable of binding to more antigens as compared to the number of its antigen-binding sites when measured in a nonhuman animal comprising a cell expressing FcRn;   (iv) an antibody that dissociates an extracellularly-bound antigen within a cell;   (v) an antibody that is bound to an antigen and internalized into a cell, and released to the outside of the cell in an antigen-free form; or   (vi) an antibody that has increased ability to eliminate an antigen in plasma.   
     
     
         31 . A method of screening for an antibody whose binding activity at a first pH is greater than that at a second pH, which comprises the steps of:
 (a) binding an antibody to an antigen-immobilized column under the condition of a first pH;   (b) eluting the antibody bound to the column at the first pH from the column under the condition of a second pH; and   (c) collecting the eluted antibody, wherein the first pH is 6.7 to 10.0 and the second pH is 4.0 to 6.5, and wherein the antibody is used as a pharmaceutical composition and is any one of the following (i) to (vi):   (i) an antibody that is superior in retention in plasma;   (ii) an antibody that is capable of binding to an antigen two or more times when measured in a nonhuman animal comprising a cell expressing FcRn;   (iii) an antibody that is capable of binding to more antigens as compared to the number of its antigen-binding sites when measured in a nonhuman animal comprising a cell expressing FcRn;   (iv) an antibody that dissociates an extracellularly-bound antigen within a cell;   (v) an antibody that is bound to an antigen and internalized into a cell, and released to the outside of the cell in an antigen-free form; or   (vi) an antibody that has increased ability to eliminate an antigen in plasma.   
     
     
         32 . A method of screening for an antibody whose binding activity at a first pH is greater than that at a second pH, which comprises the steps of:
 (a) binding an antibody library to an antigen-immobilized column under the condition of a first pH;   (b) eluting the antibody from the column under the condition of a second pH;   (c) amplifying the gene encoding the eluted antibody; and   (d) obtaining the eluted antibody, wherein the first pH is 6.7 to 10.0 and the second pH is 4.0 to 6.5, and wherein the antibody is used as a pharmaceutical composition and is any one of the following (i) to (vi):   (i) an antibody that is superior in retention in plasma;   (ii) an antibody that is capable of binding to an antigen two or more times when measured in a nonhuman animal comprising a cell expressing FcRn;   (iii) an antibody that is capable of binding to more antigens as compared to the number of its antigen-binding sites when measured in a nonhuman animal comprising a cell expressing FcRn;   (iv) an antibody that dissociates an extracellularly-bound antigen within a cell;   (v) an antibody that is bound to an antigen and internalized into a cell, and released to the outside of the cell in an antigen-free form; or   (vi) an antibody that has increased ability to eliminate an antigen in plasma.   
     
     
         33 . (canceled) 
     
     
         34 . The screening method of  claim 28 , wherein at least one or more amino acids of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody. 
     
     
         35 .- 50 . (canceled) 
     
     
         51 . The screening method of  claim 29 , wherein at least one or more amino acids of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody. 
     
     
         52 . The screening method of  claim 30 , wherein at least one or more amino acids of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody. 
     
     
         53 . The screening method of  claim 31 , wherein at least one or more amino acids of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody. 
     
     
         54 . The screening method of  claim 32 , wherein at least one or more amino acids of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody.