ANTIGEN-BINDING MOLECULES, THE ANTIGEN-BINDING ACTIVITY OF WHICH VARIES ACCORDING TO THE CONCENTRATION OF COMPOUNDS, AND LIBRARIES OF SAID MOLECULES

• US 20160304862A1
• Filed: 12/04/2014
• Published: 10/20/2016
• Est. Priority Date: 12/04/2013
• Status: Active Grant

• Current Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Original Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Inventors: TAMBA, Shigero, SHIMIZU, Shun, TATSUMI, Kanako, KADONO, Shojiro, KAWAUCHI, Hiroki, OHARA, Kazuhiro, MATSUSHITA, Masayuki, KAMIMURA, Masaki, IGAWA, Tomoyuki, EMURA, Takashi
• Granted Patent: US 10,961,530 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/248 IL-6
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/4283 against an allotypic or isotypic determinant on Ig
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

An objective of the present invention is to provide target tissue-specific antigen-binding molecules, antigen-binding molecules whose antigen-binding activity varies depending on the concentration of an unnatural compound, libraries comprising a plurality of the antigen-binding molecules which are different from one another, pharmaceutical compositions comprising the antigen-binding molecules, methods of screening for the antigen-binding molecules, and methods for producing the antigen-binding molecules. The present inventors created antigen-binding domains whose antigen-binding activity varies depending on the concentration of a small molecule compound or antigen-binding molecules containing an antigen-binding domain, and libraries comprising a plurality of the antigen-binding domains which are different from one another or antigen-binding domains, and demonstrated that the above-noted objective could be achieved by using the libraries. Various diseases originating from target tissues can be treated in a target tissue-specific manner by using the antigen-binding molecules of the present invention.

Claims

1. A library that comprises mainly:

(i) a plurality of antigen-binding domains or antigen-binding molecules comprising an antigen-binding domain, which have different sequences from one another;

or

(ii) nucleic acids that encode the plurality of antigen-binding domains or antigen-binding molecules comprising an antigen-binding domain, which have different sequences from one another;

wherein the aforementioned antigen-binding molecule domains or antigen-binding molecules are antigen-binding domains or antigen-binding molecules comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a small molecule compound.

2. The library of claim 1, which is produced by a method comprising the steps of:

(a) identifying amino acid sites that fulfill any one or more of (i) to (iii) below in antigen-binding domains whose antigen-binding activity varies depending on the concentration of a small molecule compound or in antigen-binding domains that have binding activity to a small molecule compound;

(i) one or more amino acid sites that are not involved in the binding to the small molecule compound;

(ii) one or more amino acid sites that show diversity of amino acid occurrence frequency in the antibody repertoire of the animal species to which the parent antigen-binding domain belongs; and

(iii) one or more amino acid sites that are not important for canonical structure formation; and

(b) designing a library that comprises nucleic acids encoding unmodified antigen-binding domains/molecules, and nucleic acids that encode individually a plurality of variants of the aforementioned antigen-binding domains or antigen-binding molecules comprising an antigen-binding domain which have different sequences from one another and have modifications at one or more of the amino acid sites identified in step (a).

3. The library of claim 2 which is produced by a method comprising the steps of:

(a) identifying amino acid sites that fulfill any one or more of (i) to (iii) below in antigen-binding domains whose antigen-binding activity varies depending on the concentration of a small molecule compound or in antigen-binding domains that have binding activity to a small molecule compound;

(i) one or more amino acid sites that are not involved in the binding to the small molecule compound;

(ii) one or more amino acid sites that show diversity of amino acid occurrence frequency in the antibody repertoire of the animal species to which the parent antigen-binding domain belongs; and

(iii) one or more amino acid sites that are not important for canonical structure formation;

(b) producing a plurality of variants of the aforementioned antigen-binding domains or antigen-binding molecules comprising an antigen-binding domain, which have different sequences from one another and have modifications at one or more of the amino acid sites identified in step (a);

(c) identifying one or more amino acid modifications that do not substantially change the binding activity of each of the aforementioned variants to the small molecule compound; and

(d) producing a library comprising nucleic acids that encode unmodified antigen-binding domains/molecules, and nucleic acids that encode a plurality of variants of the aforementioned antigen-binding domains or antigen-binding molecules comprising an antigen-binding domain, which have different sequences from one another and have one or more of the amino acid modifications identified in step (c).

4. The library of claim 1 produced by a method comprising the steps of:

1) contacting a library comprising a plurality of antigen-binding molecules having binding activity to a small molecule compound with the small molecule compound; and

2) concentrating from the library, nucleic acids that encode a plurality of variants of antigen-binding molecules having binding activity to the small molecule compound.

5. The library of claim 4, wherein the aforementioned antigen-binding molecules are antigen-binding molecules that comprise heavy-chain variable regions and light-chain variable regions of an antibody, and wherein the library is produced by a method comprising any one of the steps of:

1) designing a library by concentrating nucleic acids that encode a plurality of variants of antigen-binding molecules having binding activity to a small molecule compound from the library of claim 4 which comprises nucleic acids encoding one or more variants produced by modifying amino acids positioned in the heavy chain variable regions;

2) designing a library by concentrating nucleic acids that encode a plurality of variants of antigen-binding molecules having binding activity to a small molecule compound from the library of claim 4 which comprises nucleic acids encoding one or more variants produced by modifying amino acids positioned in the light chain variable regions; and

3) designing a library by combining the antigen-binding molecule-encoding nucleic acids concentrated from each of the variable region libraries of steps

     1) and

     2).

6. The library of any one of claims 1 to 5, wherein the aforementioned antigen-binding molecules are fusion polypeptides formed by fusing an antigen-binding domain with at least a portion of a virus coat protein.

7. The library of any one of claims 1 to 5, wherein the aforementioned antigen-binding molecules are antigen-binding molecules comprising antibody heavy chains and light chains, and the library further comprises a step of designing a synthetic library of the heavy chains and/or light chains.

8. The library of claim 7, wherein the antibody heavy chains and/or light chains comprise a germline-derived framework sequence.

9. The library of any one of claims 1 to 8, wherein the aforementioned small molecule compound is a target tissue-specific compound or an unnatural compound.

10. The library of any one of claims 1 to 9, wherein the aforementioned target tissue is a cancer tissue or an inflammatory tissue.

11. The library of claim 10, wherein the cancer tissue-specific compound is at least one compound selected from the group consisting of nucleosides that have a purine ring structure, amino acids and their metabolites, lipids and their metabolites, primary metabolites from sugar metabolism, and nicotinamide and its metabolites.

12. The library of any one of claims 1 to 11, wherein the small molecule compound is kynurenine, adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate.

13. The library of any one of claims 1 to 12, wherein the amino acid sites not involved in binding with the small molecule compound are sites other than any one or more of the amino acids selected from below:

H chain;

97, 100c, 101, 94, 95, 100d, 100e, 33, 50, 52, 56, 57, 58, 99, 100, 100a, 54, 55 (Kabat Numbering); and

L chain;

49, 55, 95c, 96, 95a, 95b (Kabat Numbering).

14. A method for producing an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a small molecule compound, which comprises the steps of:

(a) contacting the library of any one of claims 1 to 13 with an antigen in the absence of a small molecule compound;

(b) selecting an antigen-binding domain that does not bind to the antigen in step (a) above;

(c) contacting the antigen-binding domain selected in step (b) above with the antigen in the presence of the small molecule compound;

(d) selecting the antigen-binding domain that binds to the antigen in step (c) above;

(e) linking the polynucleotide that encodes the antigen-binding domain selected in step (d) above with a polynucleotide that encodes a polypeptide comprising an Fc region;

(f) culturing a cell introduced with a vector in which the polynucleotide obtained in step (e) above is operably linked; and

(g) collecting the antigen-binding molecule from the culture solution of the cell cultured in step (f) above.

15. A method for producing an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a small molecule compound, which comprises the steps of:

(a) contacting the library of any one of claims 1 to 13 with an antigen in the presence of a small molecule compound;

(b) collecting an antigen-binding domain by dissociating it using the small molecule compound at a lower concentration than in step (a) above;

(c) linking the polynucleotide that encodes the antigen-binding domain collected in step (b) above with a polynucleotide that encodes a polypeptide comprising an Fc region;

(d) culturing a cell introduced with a vector in which the polynucleotide obtained in step (c) above is operably linked; and

(e) collecting the antigen-binding molecule from the culture solution of the cell cultured in step (d) above.

16. The method of claim 14 or 15 for producing an antigen-binding molecule that comprises an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a small molecule compound, which further comprises the steps of:

(a) contacting the library of any one of claims 1 to 13 with a small molecule compound; and

(b) selecting antigen-binding domains collected in step (a) above.

17. The method of any one of claims 14 to 16 for producing an antigen-binding molecule, wherein the small molecule compound is kynurenine, adenosine, adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate.

18. An antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of an unnatural compound.

19. A pharmaceutical composition that comprises the antigen-binding molecule of claim 18.