Antigen-binding molecule capable of binding to two or more antigen molecules repeatedly
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. A method of screening for an antibody, the method comprising:
(a) assaying the antigen-binding activity of an antibody at pH 6.7 to pH 10.0;
(b) assaying the antigen-binding activity of the antibody at pH 4.0 to pH 6.5;
(c) determining that the antibody's antigen-binding activity at pH 6.7 to pH 10.0 is greater than its antigen-binding activity at pH 4.0 to pH 6.5;
(d) selecting the antibody based on the determination in (c); and
(e) assaying the retention in plasma of the antibody selected in step (d).
2. The method of claim 1 , wherein the determination on which the selection of (d) is based is a determination that the antibody's antigen-binding activity at pH 6.7 to pH 10.0 is at least twice as high as the antibody's antigen-binding activity at pH 4.0 to pH 6.5.
3. The method of claim 1 , wherein at least one amino acid of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody.
4. A method of screening for an antibody, the method comprising:
(a) assaying the antigen-binding activity of an antibody at pH 6.7 to pH 10.0;
(b) assaying the antigen-binding activity of the antibody at pH 4.0 to pH 6.5;
(c) determining that the antibody's antigen-binding activity at pH 6.7 to pH 10.0 is greater than its antigen-binding activity at pH 4.0 to pH 6.5;
(d) selecting the antibody based on the determination in (c); and
(e) assaying the ability of the antibody selected in step (d) to eliminate an antigen in plasma.
5. The method of claim 4 , wherein at least one amino acid of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody.
6. A method of screening for an antibody, the method comprising:
(a) binding an antibody to an antigen at 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;
(c) determining that the antibody has dissociated from the antigen under the condition of pH 4.0 to pH 6.5;
(d) selecting the antibody based on the determination in (c); and
(e) assaying the retention in plasma of the antibody obtained in step (d).
7. The method of claim 6 , wherein at least one amino acid of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody.
8. A method of screening for an antibody, the method comprising:
(a) binding an antibody to an antigen at 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;
(c) determining that the antibody has dissociated from the antigen under the condition of pH 4.0 to pH 6.5;
(d) selecting the antibody based on the determination in (c); and
(e) assaying the ability of the antibody in step (d) to eliminate an antigen in plasma.
9. The method of claim 8 , wherein at least one amino acid of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody.
10. A method of screening for an antibody whose binding activity at a first pH is greater than that at a second pH, the method comprising:
(a) binding an antibody to an antigen-immobilized column at a first pH;
(b) eluting the antibody from the column at a second pH;
(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 eluted from the column because its binding to the antigen at the first pH is greater than its binding to the antigen at the second pH; and
(d) assaying the retention in plasma of the antibody collected in step (c).
11. The method of claim 10 , wherein at least one amino acid of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody.
12. A method of screening for an antibody whose binding activity at a first pH is greater than that at a second pH, the method comprising:
(a) binding an antibody to an antigen-immobilized column at a first pH;
(b) eluting the antibody from the column at a second pH;
(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 eluted from the column because its binding to the antigen at the first pH is greater than its binding to the antigen at the second pH; and
(d) assaying the ability of the antibody collected in step (c) to eliminate an antigen in plasma.
13. The method of claim 12 , wherein at least one amino acid of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody.
14. A method of screening for an antibody whose binding activity at a first pH is greater than that at a second pH, the method comprising:
(a) binding an antibody library to an antigen-immobilized column at a first pH that is pH 6.7 to 10.0;
(b) eluting an antibody from the column at a second pH that is pH 4.0 to 6.5;
(c) amplifying nucleic acid encoding the eluted antibody;
(d) obtaining the nucleotide sequence of the nucleic acid encoding the eluted antibody;
(e) determining the amino acid sequence encoded by the nucleic acid; and
(f) producing an antibody with the amino acid sequence determined in step (e) and assaying the retention in plasma of the produced antibody.
15. The method of claim 14 , wherein at least one amino acid of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody.
16. A method of screening for an antibody whose binding activity at a first pH is greater than that at a second pH, the method comprising:
(a) binding an antibody library to an antigen-immobilized column at a first pH that is pH 6.7 to 10.0;
(b) eluting an antibody from the column at a second pH that is pH 4.0 to 6.5;
(c) amplifying nucleic acid encoding the eluted antibody;
(d) obtaining the nucleotide sequence of the nucleic acid encoding the eluted antibody;
(e) determining the amino acid sequence encoded by the nucleic acid; and
(f) producing an antibody with the amino acid sequence determined in step (e) and assaying the ability of the produced antibody to eliminate an antigen in plasma.
17. The method of claim 16 , wherein at least one amino acid of the antibody has been substituted with histidine, or at least one histidine has been inserted into the antibody.