US-12617847-B2 - Antigen-binding molecule capable of binding to plurality of antigen molecules repeatedly

US12617847B2US 12617847 B2US12617847 B2US 12617847B2US-12617847-B2

Abstract

An objective of the present invention is to provide methods for promoting antigen uptake into cells by antigen-binding molecules, methods for increasing the number of times of antigen binding by one antigen-binding molecule, methods for promoting reduction of the antigen concentration in plasma by administering antigen-binding molecules, and methods for improving the plasma retention of an antigen-binding molecule, as well as antigen-binding molecules that allow enhanced antigen uptake into cells, antigen-binding molecules having an increased number of times of antigen binding, antigen-binding molecules that can promote reduction of the antigen concentration in plasma when administered, antigen-binding molecules with improved plasma retention, pharmaceutical compositions comprising the above antigen-binding molecules, and methods for producing them. The present inventors revealed that the above objective can be achieved by using antigen-binding molecules that show calcium-dependent antigen-antibody reaction.

Inventors

Tomoyuki Igawa
Shinya Ishii
Miho Funaki
Naoka Hironiwa
Atsuhiko Maeda
Junichi Nezu
Yoshinao Ruike
Takeshi Baba
Shun Shimizu

Assignees

CHUGAI SEIYAKU KABUSHIKI KAISHA

Dates

Publication Date: 20260505
Application Date: 20231220
Priority Date: 20101130

Claims (20)

1 . A method of producing an antigen-binding molecule, the method comprising: (a) measuring the binding activity of an antigen-binding molecule to an antigen under a first calcium ion concentration that ranges from 0.1 μM to 30 μM and at a second calcium ion concentration that ranges from 100 μM to 10 mM; (b) determining that the binding activity measured at the first calcium ion concentration is lower than the binding activity measured at the second calcium ion concentration; and (c) expressing one or more polynucleotides that together encode the antigen-binding molecule, thereby producing the antigen-binding molecule.
2 . The method of claim 1 , wherein the antigen-binding molecule comprises one or more of the following characteristics: the antigen-binding molecule increases, compared to a control antibody, the rate that molecules of the antigen are taken up into cells, wherein the control antibody binds to the antigen with an antigen-binding activity that is at least as high at the first calcium ion concentration as at the second calcium ion concentration; the antigen-binding molecule carries a molecule of the antigen into a cell of a subject, releases the antigen inside the cell, returns to the subject's plasma to bind to another molecule of the antigen, and repeats the cycle at least one more time; the antigen-binding molecule reduces the concentration of the antigen in plasma of a subject more effectively than does a control antibody that binds to the antigen with an antigen-binding activity that is at least as high at the first calcium ion concentration as at the second calcium ion concentration; the antigen-binding molecule is retained in plasma of a subject longer than is a control antibody that binds to the antigen with an antigen-binding activity that is at least as high at the first calcium ion concentration as at the second calcium ion concentration.
3 . The method of claim 1 , wherein the antigen-binding molecule exhibits lower antigen-binding activity at an acidic pH than at a neutral pH.
4 . The method of claim 1 , wherein the first calcium ion concentration is an intra-endosomal concentration of ionized calcium, and the second calcium ion concentration is a plasma concentration of ionized calcium.
5 . The method of claim 1 , wherein the antigen-binding molecule comprises a light chain variable domain and a heavy chain variable domain; wherein at least four positions selected from Kabat numbering positions 30, 31, 32, 50, and 92 of the light chain variable domain are occupied by amino acids independently selected from serine, asparagine, aspartic acid, glutamic acid, histidine, and tyrosine; and wherein at least one of the at least four positions is occupied by glutamic acid or aspartic acid.
6 . The method of claim 1 , wherein the antigen-binding molecule comprises a light chain variable domain and a heavy chain variable domain, and wherein at least three positions selected from Kabat numbering positions 95, 96, 100a, and 101 of the heavy chain variable domain are occupied by amino acids independently selected from serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, histidine, and tyrosine.
7 . The method of claim 1 , wherein the antigen is human IL-6, a soluble form of human IL-6 receptor, a soluble form of human CD4, human IgA, a soluble form of human glypican 3, or human IgE.
8 . A method of producing an antigen-binding molecule, the method comprising: (a) contacting an antigen with a library comprising a plurality of antigen-binding-domain-containing proteins under a first calcium ion concentration that ranges from 100 μM to 10 mM, so that some of the proteins bind to the antigen; (b) placing proteins that bind to the antigen in step (a) under a second calcium ion concentration that ranges from 0.1 μM to 30 μM; (c) collecting proteins that dissociate from the antigen in step (b); (d) producing one or more polynucleotides that together encode an antigen-binding molecule comprising the antigen-binding domain of a protein collected in step (c); and (e) expressing the one or more polynucleotides, thereby producing the antigen-binding molecule encoded by the one or more polynucleotides.
9 . The method of claim 8 , wherein the antigen-binding molecule comprises one or more of the following characteristics: the antigen-binding molecule increases, compared to a control antibody, the rate that molecules of the antigen are taken up into cells, wherein the control antibody binds to the antigen with an antigen-binding activity that is at least as high at the first calcium ion concentration as at the second calcium ion concentration; the antigen-binding molecule carries a molecule of the antigen into a cell of a subject, releases the antigen inside the cell, returns to the subject's plasma to bind to another molecule of the antigen, and repeats the cycle at least one more time; the antigen-binding molecule reduces the concentration of the antigen in plasma of a subject more effectively than does a control antibody that binds to the antigen with an antigen-binding activity that is at least as high at the first calcium ion concentration as at the second calcium ion concentration; the antigen-binding molecule is retained in plasma of a subject longer than is a control antibody that binds to the antigen with an antigen-binding activity that is at least as high at the first calcium ion concentration as at the second calcium ion concentration.
10 . The method of claim 8 , wherein the antigen-binding molecule exhibits lower antigen-binding activity at an acidic pH than at a neutral pH.
11 . The method of claim 8 , wherein the first calcium ion concentration is an intra-endosomal concentration of ionized calcium, and the second calcium ion concentration is a plasma concentration of ionized calcium.
12 . The method of claim 8 , wherein the antigen-binding molecule comprises a light chain variable domain and a heavy chain variable domain; wherein at least four positions selected from Kabat numbering positions 30, 31, 32, 50, and 92 of the light chain variable domain are occupied by amino acids independently selected from serine, asparagine, aspartic acid, glutamic acid, histidine, and tyrosine; and wherein at least one of the at least four positions is occupied by glutamic acid or aspartic acid.
13 . The method of claim 8 , wherein the antigen-binding molecule comprises a light chain variable domain and a heavy chain variable domain, and wherein at least three positions selected from Kabat numbering positions 95, 96, 100a, and 101 of the heavy chain variable domain are occupied by amino acids independently selected from serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, histidine, and tyrosine.
14 . The method of claim 8 , wherein the antigen is human IL-6, a soluble form of human IL-6 receptor, a soluble form of human CD4, human IgA, a soluble form of human glypican 3, or human IgE.
15 . A method of producing an antigen-binding molecule, the method comprising: (a) contacting an antigen with a library comprising a plurality of antigen-binding-domain-containing proteins under a first calcium ion concentration that ranges from 0.1 μM to 30 μM, so that some of the proteins bind to the antigen; (b) collecting proteins that do not bind to the antigen in step (a); (c) placing proteins collected in step (b) in contact with the antigen under a second calcium ion concentration that ranges from 100 μM to 10 mM; (d) collecting proteins that are bound to the antigen in step (c); (e) producing one or more polynucleotides that together encode an antigen-binding molecule comprising the antigen-binding domain of a protein collected in step (d); and (f) expressing the one or more polynucleotides, thereby producing the antigen-binding molecule encoded by the one or more polynucleotides.
16 . The method of claim 15 , wherein the antigen-binding molecule comprises one or more of the following characteristics: the antigen-binding molecule increases, compared to a control antibody, the rate that molecules of the antigen are taken up into cells, wherein the control antibody binds to the antigen with an antigen-binding activity that is at least as high at the first calcium ion concentration as at the second calcium ion concentration; the antigen-binding molecule carries a molecule of the antigen into a cell of a subject, releases the antigen inside the cell, returns to the subject's plasma to bind to another molecule of the antigen, and repeats the cycle at least one more time; the antigen-binding molecule reduces the concentration of the antigen in plasma of a subject more effectively than does a control antibody that binds to the antigen with an antigen-binding activity that is at least as high at the first calcium ion concentration as at the second calcium ion concentration; the antigen-binding molecule is retained in plasma of a subject longer than is a control antibody that binds to the antigen with an antigen-binding activity that is at least as high at the first calcium ion concentration as at the second calcium ion concentration.
17 . The method of claim 15 , wherein the antigen-binding molecule exhibits lower antigen-binding activity at an acidic pH than at a neutral pH.
18 . The method of claim 15 , wherein the first calcium ion concentration is an intra-endosomal concentration of ionized calcium, and the second calcium ion concentration is a plasma concentration of ionized calcium.
19 . The method of claim 15 , wherein the antigen-binding molecule comprises a light chain variable domain and a heavy chain variable domain; wherein at least four positions selected from Kabat numbering positions 30, 31, 32, 50, and 92 of the light chain variable domain are occupied by amino acids independently selected from serine, asparagine, aspartic acid, glutamic acid, histidine, and tyrosine; and wherein at least one of the at least four positions is occupied by glutamic acid or aspartic acid.
20 . The method of claim 15 , wherein the antigen-binding molecule comprises a light chain variable domain and a heavy chain variable domain, and wherein at least three positions selected from Kabat numbering positions 95, 96, 100a, and 101 of the heavy chain variable domain are occupied by amino acids independently selected from serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, histidine, and tyrosine.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional of U.S. patent application Ser. No. 15/988,348, filed on May 24, 2018 (now U.S. Pat. No. 11,891,434), which is a divisional application of U.S. patent application Ser. No. 13/990,158, now abandoned, filed on Mar. 28, 2014, which is the National Stage of International Patent Application No. PCT/JP2011/077619, filed on Nov. 30, 2011, which claims the benefit of Japanese Patent Application No. 2010-266121, filed on Nov. 30, 2010, and Japanese Patent Application No. 2011-217886, filed on Sep. 30, 2011. The entire content of parent application Ser. No. 15/988,348 is hereby incorporated by reference. SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically as an XML file named 38856-0226003_SL_ST26.xml. The XML file, created on Dec. 11, 2023, is 132,225 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. BACKGROUND ART Antibodies are drawing attention as pharmaceuticals as they are highly stable in plasma and have few side effects. At present, a number of IgG-type antibody pharmaceuticals are available on the market and many antibody pharmaceuticals are currently under development (Non-patent Documents 1 and 2). Meanwhile, various technologies applicable to second-generation antibody pharmaceuticals have been reported, including those that enhance effector function, antigen-binding ability, pharmacokinetics, and stability, and those that reduce the risk of immunogenicity (Non-patent Document 3). In general, the requisite dose of an antibody pharmaceutical is very high. This in turn has led to problems such as high production cost, as well as the difficulty in producing subcutaneous formulations. In theory, the dose of an antibody pharmaceutical may be reduced by improving antibody pharmacokinetics or improving the affinity between antibodies and antigens. The literature has reported methods for improving antibody pharmacokinetics using artificial substitution of amino acids in constant regions (Non-patent Documents 4 and 5). Similarly, affinity maturation has been reported as a technology for enhancing antigen-binding ability or antigen-neutralizing activity (Non-patent Document 6). This technology enables enhancement of antigen-binding activity by introducing amino acid mutations into the CDR region of a variable region or such. The enhancement of antigen-binding ability enables improvement of in vitro biological activity or reduction of dosage, and further enables improvement of in vivo efficacy (Non-patent Document 7). Meanwhile, the antigen-neutralizing capacity of a single antibody molecule depends on its affinity. By increasing the affinity, an antigen can be neutralized by a smaller amount of an antibody. Various methods can be used to enhance antibody affinity (Non-patent Document 6). Furthermore, if the affinity could be made infinite by covalently binding the antibody to the antigen, a single antibody molecule could neutralize one antigen molecule (a divalent antibody can neutralize two antigen molecules). However, the stoichiometric neutralization of one antibody against one antigen (one divalent antibody against two antigens) is the limit of pre-existing methods, and thus it was impossible to completely neutralize antigen with an amount of antibody smaller than the amount of antigen. In other words, the affinity-enhancing effect has a limit (Non-Patent Document 9). To prolong the neutralization effect of a neutralizing antibody for a certain period, the antibody must be administered at a dose higher than the amount of antigen produced in the body during the same period. Therefore, with just the above-described improvement of antibody pharmacokinetics or affinity maturation technology, there were limitations when it comes to reduction of the required antibody dose. Accordingly, in order to sustain antibody's antigen-neutralizing effect for a target period with an amount of the antibody smaller than the amount of antigen, a single antibody must neutralize multiple antigens. An antibody that binds to an antigen in a pH-dependent manner has recently been reported as a novel method for achieving the above objective (Patent Document 1). The antibodies with pH-dependent antigen binding, which strongly bind to an antigen under the neutral conditions in plasma and dissociate from the antigen under acidic conditions in the endosome, can dissociate from the antigen in the endosome. When an antibody with pH-dependent antigen binding dissociates from the antigen is recycled to the plasma by FcRn, it can bind to another antigen again. Thus, a single antibody can repeatedly bind to a number of antigens. In addition, plasma retention of the antigen is very short as compared to antibodies recycled via FcRn binding. When an antibody with long plasma retention binds to such an antigen with a short plasma retention, the plasma retention time of the antigen-antib