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 . An antigen-binding molecule having a KD (pH5.8)/KD (pH7.4) value, defined as the ratio of KD for the antigen at pH 5.8 and KD for the antigen at pH 7.4, of 2 or higher.
2 . The antigen-binding molecule of claim 1 , wherein the KD (pH5.8)/KD (pH7.4) value is 10 or higher.
3 . The antigen-binding molecule of claim 1 , wherein the KD (pH5.8)/KD (pH7.4) value is 40 or higher.
4 . The antigen-binding molecule of claim 1 , wherein at least one amino acid of the antigen-binding molecule has been substituted with histidine, or at least one histidine has been inserted into the antigen-binding molecule.
5 . The antigen-binding molecule of claim 1 , wherein the antigen-binding molecule has an antagonistic activity.
6 . The antigen-binding molecule of claim 1 , wherein the antigen-binding molecule binds to a membrane antigen or a soluble antigen.
7 . The antigen-binding molecule of claim 1 , wherein the antigen-binding molecule is an antibody.
8 . A pharmaceutical composition comprising the antigen-binding molecule of claim 1 .
9 . A method for improving the pharmacokinetics of an antigen-binding molecule by impairing the antigen-binding activity of the antigen-binding molecule at pH 5.8 as compared to that at pH 7.4.
10 . A method for increasing the number of times of antigen-binding for an antigen-binding molecule by impairing the antigen-binding activity of the antigen-binding molecule at pH 5.8 as compared to that at pH 7.4.
11 . A method for increasing the number of antigens that can be bound by an antigen-binding molecule by impairing the antigen-binding activity of the antigen-binding molecule at pH 5.8 as compared to that at pH 7.4.
12 . A method for dissociating within a cell an antigen from an extracellularly-bound antigen-binding molecule by impairing the antigen-binding activity of the antigen-binding molecule at pH 5.8 as compared to that at pH 7.4.
13 . A method for releasing an antigen-binding molecule, which has been bound to an antigen and internalized into a cell, in an antigen-free form to the outside of the cell by impairing the antigen-binding activity of the antigen-binding molecule at pH 5.8 as compared to that at pH 7.4.
14 . A method for increasing the ability of an antigen-binding molecule to eliminate an antigen in plasma by impairing the antigen-binding activity of the antigen-binding molecule at pH 5.8 as compared to that at pH 7.4.
15 . The method of claim 10 , wherein the KD (pH5.8)/KD (pH7.4) value, defined as the ratio of KD for the antigen at pH 5.8 and KD for the antigen at pH 7.4, is 2 or higher.
16 . The method of claim 10 , wherein the KD (pH5.8)/KD (pH7.4) value is 10 or higher.
17 . The method of claim 10 , wherein the KD (pH5.8)/KD (pH7.4) value is 40 or higher.
18 . A method for improving the pharmacokinetics of an antigen-binding molecule by substituting at least one amino acid of the antigen-binding molecule with histidine, or inserting at least one histidine into the antigen-binding molecule.
19 . A method for increasing the number of times of antigen-binding for an antigen-binding molecule by substituting at least one amino acid of the antigen-binding molecule with histidine, or inserting at least one histidine into the antigen-binding molecule.
20 . A method for increasing the number of antigens that can be bound by an antigen-binding molecule by substituting at least one amino acid of the antigen-binding molecule with histidine, or inserting at least one histidine into the antigen-binding molecule.
21 . A method for dissociating within a cell an antigen from an extracellularly-bound antigen-binding molecule by substituting at least one amino acid of the antigen-binding molecule with histidine, or inserting at least one histidine into the antigen-binding molecule.
22 . A method for releasing an antigen-binding molecule, which has been bound to an antigen and internalized into a cell, in an antigen-free form to the outside of the cell, by substituting at least one amino acid of the antigen-binding molecule with histidine, or inserting at least one histidine into the antigen-binding molecule.
23 . A method for increasing the ability of an antigen-binding molecule to eliminate an antigen in plasma by substituting at least one amino acid of the antigen-binding molecule with histidine, or inserting at least one histidine into the antigen-binding molecule.
24 . The method of claim 19 , wherein the histidine substitution or insertion increases the KD (pH5.8)/KD (pH7.4) value, defined as the ratio of the antigen-binding activity at pH 5.8 and the antigen-binding activity at pH 7.4, as compared to the KD (pH5.8)/KD (pH7.4) value before the histidine substitution or insertion.
25 . The method of claim 10 , wherein the antigen-binding molecule has an antagonistic activity.
26 . The method of claim 10 , wherein the antigen-binding molecule binds to a membrane antigen or a soluble antigen.
27 . The method of claim 10 , wherein the antigen-binding molecule is an antibody.
28 . A method of screening for an antigen-binding molecule, which comprises the steps of:
(a) determining the antigen-binding activity of an antigen-binding molecule at pH 6.7 to pH 10.0;
(b) determining the antigen-binding activity of the antigen-binding molecule at pH 4.0 to pH 6.5; and
(c) selecting an antigen-binding molecule 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.
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 antigen-binding molecule, which comprises the steps of:
(a) binding an antigen-binding molecule to an antigen under a condition of pH 6.7 to pH 10.0;
(b) placing the antigen-binding molecule that bound to the antigen of (a) under a condition of pH 4.0 to pH 6.5; and
(c) obtaining an antigen-binding molecule that dissociated under the condition of pH 4.0 to pH 6.5.
31 . A method of screening for an antigen-binding molecule whose binding activity at a first pH is greater than that at a second pH, which comprises the steps of:
(a) binding an antigen-binding molecule to an antigen-immobilized column under the condition of a first pH;
(b) eluting the antigen-binding molecule that had bound to the column at the first pH from the column under the condition of a second pH; and
(c) collecting the eluted antigen-binding molecule.
32 . A method of screening for an antigen-binding molecule whose binding activity at a first pH is greater than that at a second pH, which comprises the steps of:
(a) binding an antigen-binding molecule library to an antigen-immobilized column under the condition of a first pH;
(b) eluting the antigen-binding molecule from the column under the condition of a second pH;
(c) amplifying the gene encoding the eluted antigen-binding molecule; and
(d) obtaining the eluted antigen-binding molecule.
33 . The screening method of claim 31 , wherein the first pH is 6.7 to 10.0 and the second pH is 4.0 to 6.5.
34 . The screening method of claim 28 , wherein at least one or more amino acids of the antigen-binding molecule has been substituted with histidine, or at least one histidine has been inserted into the antigen-binding molecule.
35 . The screening method of claim 28 , for obtaining an antigen-binding molecule that is superior in retention in the plasma.
36 . The screening method of claim 28 , for obtaining an antigen-binding molecule that is capable of binding to an antigen two or more times.
37 . The screening method of claim 28 , for obtaining an antigen-binding molecule that is capable of binding to more antigens as compared to the number of its antigen-binding sites.
38 . The screening method of claim 28 , for obtaining an antigen-binding molecule that dissociates an extracellularly-bound antigen within a cell.
39 . The screening method of claim 28 , for obtaining an antigen-binding molecule that is bound to an antigen and internalized into a cell, and released to the outside of the cell in an antigen-free form.
40 . The screening method of claim 28 , for obtaining an antigen-binding molecule that has increased ability to eliminate an antigen in plasma.
41 . The screening method of claim 28 , wherein the antigen-binding molecule is used as a pharmaceutical composition.
42 . The screening method of claim 28 , wherein the antigen-binding molecule is an antibody.
43 . A method for producing an antigen-binding molecule, which comprises the steps of:
(a) determining the antigen-binding activity of an antigen-binding molecule at pH 6.7 to pH 10.0;
(b) determining the antigen-binding activity of the antigen-binding molecule at pH 4.0 to pH 6.5;
(c) selecting the antigen-binding molecule whose antigen-binding activity at pH 6.7 to pH 10.0 is greater than that at pH 4.0 to pH 6.5;
(d) obtaining the gene encoding the antigen-binding molecule selected in (c); and
(e) producing the antigen-binding molecule using the gene obtained in (d).
44 . A method for producing an antigen-binding molecule, which comprises the steps of:
(a) binding an antigen-binding molecule to an antigen under a condition of pH 6.7 to pH 10.0;
(b) allowing the antigen-binding molecule bound to the antigen of (a) to stand under a condition of pH 4.0 to pH 6.5;
(c) collecting the antigen-binding molecule that dissociated under the condition of pH 4.0 to pH 6.5;
(d) obtaining the gene encoding the antigen-binding molecule obtained in (c); and
(e) producing the antigen-binding molecule using the gene obtained in (d).
45 . A method for producing an antigen-binding molecule whose binding activity at a first pH is greater than that at a second pH, which comprises the steps of:
(a) binding an antigen-binding molecule to an antigen-immobilized column under the first pH condition;
(b) eluting the antigen-binding molecule, which is bound to the column at the first pH, from the column under a second pH condition;
(c) collecting the eluted antigen-binding molecule;
(d) obtaining the gene encoding the antigen-binding molecule obtained in (c); and
(e) producing the antigen-binding molecule using the gene obtained in (d).
46 . A method for producing an antigen-binding molecule whose binding activity at a first pH is greater than that at a second pH, which comprises the steps of:
(a) binding an antigen-binding molecule library to an antigen-immobilized column under the first pH condition;
(b) eluting the antigen-binding molecule from the column under the second pH condition;
(c) amplifying the gene encoding the eluted antigen-binding molecule;
(d) collecting the eluted antigen-binding molecule;
(e) obtaining the gene encoding the antigen-binding molecule collected in (d); and
(f) producing the antigen-binding molecule using the gene obtained in (e).
47 . The production method of claim 45 , wherein the first pH is 6.7 to 10.0 and the second pH is 4.0 to 6.5.
48 . The production method of claim 43 , which further comprises the step of substituting at least one amino acid of the antigen-binding molecule with histidine, or inserting at least one histidine into the antigen-binding molecule.
49 . The production method of claim 43 , wherein the antigen-binding molecule is an antibody.
50 . A pharmaceutical composition comprising an antigen-binding molecule produced by the production method of claim 43 .
51 . The method of claim 19 , wherein the antigen-binding molecule has an antagonistic activity.
52 . The method of claim 19 , wherein the antigen-binding molecule binds to a membrane antigen or a soluble antigen.
53 . The method of claim 19 , wherein the antigen-binding molecule is an antibody.
54 . The screening method of claim 31 , wherein at least one or more amino acids of the antigen-binding molecule has been substituted with histidine, or at least one histidine has been inserted into the antigen-binding molecule.
55 . The screening method of claim 31 , for obtaining an antigen-binding molecule that is superior in retention in the plasma.
56 . The screening method of claim 31 , for obtaining an antigen-binding molecule that is capable of binding to an antigen two or more times.
57 . The screening method of claim 31 , for obtaining an antigen-binding molecule that is capable of binding to more antigens as compared to the number of its antigen-binding sites.
58 . The screening method of claim 31 , for obtaining an antigen-binding molecule that dissociates an extracellularly-bound antigen within a cell.
59 . The screening method of claim 31 , for obtaining an antigen-binding molecule that is bound to an antigen and internalized into a cell, and released to the outside of the cell in an antigen-free form.
60 . The screening method of claim 31 , for obtaining an antigen-binding molecule that has increased ability to eliminate an antigen in plasma.
61 . The screening method of claim 31 , wherein the antigen-binding molecule is used as a pharmaceutical composition.
62 . The screening method of claim 31 , wherein the antigen-binding molecule is an antibody.
63 . The production method of claim 45 , which further comprises the step of substituting at least one amino acid of the antigen-binding molecule with histidine, or inserting at least one histidine into the antigen-binding molecule.
64 . The production method of claim 45 , wherein the antigen-binding molecule is an antibody.
65 . A pharmaceutical composition comprising an antigen-binding molecule produced by the production method of claim 45 .