Antigen-binding molecule containing two antigen-binding domains that are linked to each other

Patent metadata

Field	Value
Publication number	US-12435137-B2
Application number	US-201917264388-A
Country	US
Kind code	B2
Filing date	Aug 2, 2019
Priority date	Aug 3, 2018
Publication date	Oct 7, 2025
Grant date	Oct 7, 2025

Abstract

In a non-limiting embodiment, the present invention relates to antigen-binding molecules comprising two or more antigen-binding domains which are linked with each other. In a non-limiting embodiment, the antigen-binding molecules of the present disclosure have activity of holding two or more antigen molecules at spatially close positions, activity of regulating interaction between two or more antigen molecules, activity of regulating activation of two or more antigen molecules which are activated by association with each other, resistance to protease cleavage, or such.

First claim

The invention claimed is:

1. An antigen-binding molecule comprising a first antigen-binding domain crosslinked with a second antigen-binding domain,
wherein the first antigen-binding domain and the second antigen-binding domain domains comprise a hinge region,
wherein the first and second antigen-binding domains each comprise an antibody fragment which binds to a particular antigen,
wherein the first and second antigen-binding domains are linked with each other by two or more bonds between amino acid residues in the first and second antigen binding domains,
wherein at least one of the bonds is a disulfide bond, and
wherein at least one amino acid residue from which the two or more bonds originate is present within a CH1 region and/or within a CL region of the antibody fragment of the first and second antigen-binding domains and at least one amino acid residue from which the bonds originate is present within a hinge region, wherein at least one of the bonds linking the two antigen-binding domains is formed by:
linking an amino acid residue in a CH1 region of the first antigen-binding domain with an amino acid residue in a CH1 region of the second antigen-binding domain,
wherein at least one of the bonds linking the two antigen-binding domains is formed by linking any two amino acid residues selected from the group consisting of positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, and 201 to 214, according to EU numbering, in a CH1 region of the first antigen-binding domain and a CH1 region of the second antigen binding domain,
linking an amino acid residue in a CL region of the first antigen-binding domain with an amino acid residue in a CL region of the second antigen-binding domain,
wherein at least one of the bonds linking the two antigen-binding domains is formed by linking any two amino acid residues selected from the group consisting of positions 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212, and 213, according to Kabat numbering, in a CL region of the first antigen-binding domain and a CL region of the second antigen binding domain, or
linking an amino acid residue in a CH1 region of the first antigen-binding domain with an amino acid residue in a CL region of the second antigen-binding domain,
wherein the amino acid residue in a CH1 region is selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197, according to EU numbering, and the amino acid residue in a CL region is selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128, according to Kabat numbering, and
wherein said antigen-binding molecule has increased resistance to protease cleavage as compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule only in that the control antigen-binding molecule has one less bond between the two antigen-binding domains.

2. The antigen-binding molecule of claim 1 , wherein the antibody fragment of the first and second antigen-binding domains is a Fab, Fab′, or scFab.

3. The antigen-binding molecule of claim 1 , wherein both the first and the second antigen-binding domains comprise an Fc region.

4. The antigen-binding molecule of claim 1 , which has activity of regulating interaction between two antigen molecules.

5. A pharmaceutical composition comprising the antigen-binding molecule of claim 1 and a pharmaceutically acceptable carrier.

6. A method for regulating interaction between two antigen molecules, comprising:
(a) providing an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first and second antigen-binding domains comprise a hinge region and each of the first and second antigen-binding domains comprise an antibody fragment which binds to a particular antigen,
(b) adding to the antigen-binding molecule at least one bond which links the first and second antigen-binding domains, wherein the first and second antigen-binding domains are linked with each other by two or more bonds, wherein at least one of the bonds is a disulfide bond,
wherein at least one amino acid residue from which a bond between antigen-binding domains originates is present within a CH1 region and/or within a CL region of the antibody fragment of the first and second antigen-binding domains and at least one amino acid residue from which a bond between antigen-binding domains originates is present within a hinge region;
wherein at least one of the bonds linking the first and second antigen-binding domains is formed by:

linking an amino acid residue in a CH1 region of the first antigen-binding domain with an amino acid residue in a CH1 region of the second antigen-binding domain,
wherein at least one of the bonds linking the two antigen-binding domains is formed by linking any two amino acid residues selected from the group consisting of positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, and 201 to 214, according to EU numbering, in a CH1 region of the first antigen-binding domain and a CH1 region of the second antigen binding domain,
linking an amino acid residue in a CL region of the first antigen-binding domain with an amino acid residue in a CL region of the second antigen-binding domain,
wherein at least one of the bonds linking the two antigen-binding domains is formed by linking any two amino acid residues selected from the group consisting of positions 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212, and 213, according to Kabat numbering, in a CL region of the first antigen-binding domain and a CL region of the second antigen-binding domain, or
linking an amino acid residue in a CH1 region of the first antigen-binding domain with an amino acid residue in a CL region of the second antigen-binding domain,
wherein the amino acid residue in a CH1 region is selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196, or 197, according to EU numbering, and the amino acid residue in a CL region is selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128, according to Kabat numbering, and
(c) contacting the antigen-binding molecule produced in (b) with the two antigen molecules, and
wherein said antigen-binding molecule has increased resistance to protease cleavage as compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule only in that the control antigen-binding molecule has one less bond between the two antigen-binding domains.

7. A method for producing an antigen-binding molecule which has activity of regulating interaction between two antigen molecules, comprising:
(a) providing a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein the first and second antigen-binding domains comprise a hinge region and the first and second antigen-binding domains each comprises an antibody fragment which binds to a particular antigen,
(b) introducing a mutation into nucleic acids encoding the first and second antigen-binding domains such that at least one bond linking the first and second antigen-binding domains is added, wherein the first antigen-binding domain and the second antigen-binding domain are linked with each other via two or more bonds, wherein at least one of the bonds is a disulfide bond;
wherein at least one amino acid residue from which a bond between antigen-binding domains originates is p

Assignees

• Chugai Pharmaceutical Co Ltd

Inventors

• Shiraiwa Hirotake
• Kawa Tatsuya

Classifications

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C07K2317/76

  Antagonist effect on antigen, e.g. neutralization or inhibition of binding · CPC title

• C07K2317/75

  Agonist effect on antigen · CPC title

• C07K2317/569

  Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® · CPC title

• C07K2317/55

  Fab or Fab' · CPC title