Antigen-binding molecules and libraries whose antigen-binding ability changes depending on the concentration of the compound.

Abstract

To provide an antigen-binding molecule specific to a target tissue, an antigen-binding molecule whose antigen-binding activity changes dependently on concentration of an unnatural compounds, a library including a plurality of different antigen-binding molecules, a pharmaceutical composition containing the antigen-binding molecule, a method for screening the antigen-binding molecule, a producing method thereof, and like.SOLUTION: It was found that above problem may be solved by generating an antigen-binding domain or an antigen-binding molecule comprising the antigen-binding domain whose binding activity to an antigen changes dependently on concentration of low-molecular-weight compounds, creating a plurality of different antigen-binding domains and a library including the antigen-binding domains, and using the library. By using the antigen-binding molecule of the present invention, various diseases caused by target tissue can be treated specifically for the target tissue.SELECTED DRAWING: None

Claims

1. (i) Multiple antigen-binding domains with different sequences from each other, or an antigen-binding molecule containing antigen-binding domains; or (ii) Nucleic acids encoding a plurality of antigen-binding domains or an antigen-binding molecule containing antigen-binding domains, each having different sequences from the others. A library for obtaining antigen-binding domains or antigen-binding molecules containing antigen-binding domains, the antigen-binding activity of which changes depending on the concentration of the low-molecular-weight compound, which mainly contains the following: The aforementioned plurality of antigen-binding domains or antigen-binding molecules containing antigen-binding domains are antigen-binding domains or antigen-binding molecules whose binding activity to the antigen changes depending on the concentration of the low-molecular-weight compound. The antigen-binding domain includes an antibody heavy chain variable region and an antibody light chain variable region. The low molecular weight compound is adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate. The antibody heavy chain variable region is (a1) CDR1, CDR2, and CDR3 included in the heavy chain variable region indicated by Sequence ID: 85; or (a2) CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 85, in which one or more amino acid residues selected from positions 31, 32, 53, 54, 55, 56, 57, 59, 61, 62, 65, 96, 97, 98, 100, 100a, 101 and 102 (Kabat numbering) of the heavy chain variable region of SEQ ID NO: 85 have been modified. including and The aforementioned antibody light chain variable region is (b1) CDR1, CDR2, and CDR3 included in the light chain variable region indicated by Sequence ID: 86; or (b2) CDR1, CDR2 and CDR3 included in the light chain variable region represented by SEQ ID NO: 86, in which one or more amino acid residues selected from positions 28, 29, 32, 93, 94, 95, 95a, 95b and 95c (Kabat numbering) of the light chain variable region represented by SEQ ID NO: 86 have been modified. including, Library.

2. The antibody heavy chain variable region is (c1) Heavy chain framework included in the heavy chain variable region indicated by Sequence ID: 85; or (c2) Heavy chain framework included in the heavy chain variable region shown in Sequence ID: 30; The library according to claim 1, further comprising:

3. The aforementioned antibody light chain variable region is (d1) Light chain framework included in the light chain variable region indicated by Sequence ID: 86; or (d2) Light chain framework included in the light chain variable region indicated by sequence number 31; The library according to claim 1 or claim 2, further comprising:

4. The above (i) or (ii) is 10 different independent clones of the sequence in the library. -6 A library according to any one of claims 1 to 3, wherein the proportion is between % and 80%.

5. The library according to any one of claims 1 to 4, wherein the antigen-binding molecule of the library is the structure of a Fab fragment or the structure of a Fab fragment fused with a viral coat protein or a part of a viral coat protein.

6. The library according to any one of claims 1 to 4, wherein the antigen-binding molecule of the library is a single-chain Fv (scFv) structure or a structure of scFv fused with a viral coat protein or a part of a viral coat protein.

7. (a) A step of contacting the library according to any one of claims 1 to 6 with an antigen in the absence of a low molecular weight compound; (b) A step of selecting an antigen-binding domain that does not bind to the antigen in step (a); (c) A step of contacting the antigen-binding domain selected in step (b) with the antigen in the presence of the low molecular weight compound; (d) A step of selecting an antigen-binding domain that binds to the antigen in step (c); (e) A step of ligating a polynucleotide encoding an antigen-binding domain selected in step (d) to a polynucleotide encoding a polypeptide containing an Fc region; (f) A step of culturing cells into which a vector in which the polynucleotides obtained in step (e) are operably linked has been introduced; and (g) A step of recovering antigen-binding molecules from the culture medium of the cells cultured in step (f) above, Includes, The low molecular weight compound is adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate. A method for producing an antigen-binding molecule containing an antigen-binding domain.

8. (a) A step of contacting an antigen with a library according to any one of claims 1 to 6 in the presence of a low molecular weight compound; (b) A step of dissociating and recovering the antigen-binding domain with a low-molecular-weight compound at a lower concentration than in step (a); (c) A step of ligating the polynucleotide encoding the antigen-binding domain recovered in step (b) to a polynucleotide encoding a polypeptide containing an Fc region; (d) A step of culturing cells into which a vector in which the polynucleotides obtained in step (c) are operably linked has been introduced; and (e) A step of recovering antigen-binding molecules from the culture medium of the cells cultured in step (d), Includes, The low molecular weight compound is adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate. A method for producing an antigen-binding molecule containing an antigen-binding domain.

9. (a) the step of contacting the library according to any one of claims 1 to 6 with the low molecular weight compound; and (b) A step of selecting the antigen-binding domain bound to the low molecular weight compound in step (a), The method according to claim 7 or claim 8, further comprising:

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