ANTIBODY-IMMOBILIZED CARRIER, METHOD OF PRODUCING ANTIBODY-IMMOBILIZED CARRIER, AND USE OF SAID ANTIBODY-IMMOBILIZED CARRIER

• US 20120309943A1
• Filed: 02/15/2011
• Published: 12/06/2012
• Est. Priority Date: 02/16/2010
• Status: Active Grant

• Current Assignee: Denka Company Limited, National University Corporation Tokyo Institute of Technology
• Original Assignee: Denka Seiken Co., Ltd. (Denka Company Limited), National University Corporation Tokyo Institute of Technology
• Inventors: Kumada, Yoichi, Kishimoto, Michimasa, Hamasaki, Kyoko, Nakagawa, Aya
• Granted Patent: US 8,883,985 B2
• Time in Patent Office: Days
• US Class Current: 530/389.1
• 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 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/249 Interferons
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/303 Liver or Pancreas
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 17/00 Carrier-bound or immobilised peptides carrier-bound or immobilised enzymes C12N11/00; Preparation thereof
• 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 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/55 Fab or Fab'

Abstract

The present invention provides an antibody-immobilized carrier that can be used in antibody screening, a method of producing the antibody-immobilized carrier, and use of the antibody-immobilized carrier. Efficient antibody screening can be carried out particularly by an antibody-immobilized carrier including two or more antibody immobilized regions onto each of which a heavy-chain low-molecular-weight antibody and a light-chain low-molecular-weight antibody are separately immobilized, the two or more antibody immobilized regions each being included in an independent manner, the heavy-chain low-molecular-weight antibody including a heavy-chain variable region, the light-chain low-molecular-weight antibody including a light-chain variable region, the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody each being derived from an antibody recognizing a different antigen.

Claims

1. An antibody-immobilized carrier comprising at least one antibody immobilized region where a heavy-chain low-molecular-weight antibody and a light-chain low-molecular-weight antibody are separately immobilized,

the at least one antibody immobilized region being included in an independent manner,

the heavy-chain low-molecular-weight antibody including a heavy-chain variable region,

the light-chain low-molecular-weight antibody including a light-chain variable region.

2. The antibody-immobilized carrier as set forth in claim 1, wherein:

each of the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody is separately immobilized onto a carrier via a carrier binding peptide that binds to a material of a carrier surface; and

the carrier binding peptide is provided to a C-terminus side of the heavy-chain variable region in the heavy-chain low-molecular-weight antibody or to a C-terminus side of the light-chain variable region in the light-chain low-molecular-weight antibody.

3. The antibody-immobilized carrier as set forth in claim 2, wherein:

the material of the carrier surface is plastic resin having been hydrophilized by property modification, the plastic resin being polystyrene, polycarbonate, polypropylene, polyethylene, polydimethylsiloxane or polymethyl methacrylate.

4. The antibody-immobilized carrier as set forth in claim 2, wherein:

the carrier binding peptide is a peptide that binds to hydrophilic polystyrene, hydrophilic polycarbonate, hydrophilic polypropylene, hydrophilic polyethylene, hydrophilic polydimethylsiloxane or hydrophilic polymethyl methacrylate.

5. The antibody-immobilized carrier as set forth in claim 1, wherein:

the heavy-chain low-molecular-weight antibody is a heavy-chain low-molecular-weight antibody consisting of the heavy-chain variable region or a heavy-chain low-molecular-weight antibody consisting of a heavy-chain variable region and a first heavy-chain constant region.

6. The antibody-immobilized carrier as set forth in claim 1, wherein:

the light-chain low-molecular-weight antibody is a light-chain low-molecular-weight antibody consisting of the light-chain variable region or a light-chain low-molecular-weight antibody consisting of a light-chain variable region and a light-chain constant region.

14. An antibody screening method comprising the step of:

screening a heavy-chain low-molecular-weight antibody and/or a light-chain low-molecular-weight antibody each recognizing a specific antigen, by using the antibody-immobilized carrier as set forth in claim 1.

15. A method of screening a human antibody recognizing a specific antigen by using the antibody-immobilized carrier as set forth in claim 1, the screening being carried out by using a chimeric antibody or a humanized antibody each recognizing the specific antigen,

the heavy-chain low-molecular-weight antibody being immobilized onto the at least one antibody immobilized region and including a heavy-chain variable region derived from the chimeric antibody or the humanized antibody,

the light-chain low-molecular-weight antibody being a light-chain low-molecular-weight antibody that is immobilized onto the at least one antibody immobilized region and that includes a light-chain variable region derived from a random human antibody,

the method comprising the steps of:

(i) putting the specific antigen in contact with the antibody-immobilized carrier;

(ii) detecting an antibody immobilized region recognizing the specific antigen on the antibody-immobilized carrier; and

(iii) determining a light-chain low-molecular-weight antibody immobilized on the antibody immobilized region detected in the step (ii), as a candidate for a light-chain variable region of the human antibody recognizing the specific antigen.

16. The method of screening as set forth in claim 15, further comprising the steps of:

(iv) putting the specific antigen in contact with another antibody-immobilized carrier including another antibody immobilized region onto which (a) the light-chain low-molecular-weight antibody determined as the candidate in the step (iii) and (b) a heavy-chain low-molecular-weight antibody including a heavy-chain variable region derived from a random human antibody are immobilized;

(v) detecting an antibody immobilized region recognizing the specific antibody on the another antibody-immobilized carrier; and

(vi) determining a heavy-chain low-molecular-weight antibody immobilized onto the antibody immobilized region detected in the step (v), as a candidate for a heavy-chain variable region of the human antibody recognizing the specific antigen.

17. A method of screening a human antibody recognizing a specific antigen by using the antibody-immobilized carrier as set forth in claim 1, the screening being carried out by using a chimeric antibody or a humanized antibody each recognizing the specific antigen,

the light-chain low-molecular-weight antibody being immobilized onto the at least one antibody immobilized region and including a light-chain variable region derived from the chimeric antibody or the humanized antibody,

the heavy-chain low-molecular-weight antibody being a heavy-chain low-molecular-weight antibody that is immobilized onto the at least one antibody immobilized region and that includes a heavy-chain variable region derived from a random human antibody,

the method comprising the steps of:

(i) putting the specific antigen in contact with the antibody-immobilized carrier;

(ii) detecting an antibody immobilized region recognizing the specific antigen on the antibody-immobilized carrier; and

(iii) determining a heavy-chain low-molecular-weight antibody immobilized on the antibody immobilized region detected in the step (ii), as a candidate for a heavy chain variable region of the human antibody recognizing the specific antigen.

18. The method of screening as set forth in claim 17, further comprising the steps of:

(iv) putting the specific antigen in contact with another antibody-immobilized carrier including another antibody immobilized region onto which (a) the heavy-chain low-molecular-weight antibody determined as the candidate in the step (iii) and (b) a light-chain low-molecular-weight antibody including a light-chain variable region derived from a random human antibody are immobilized;

(v) detecting an antibody immobilized region recognizing the specific antibody on the another antibody-immobilized carrier; and

(vi) determining a light-chain low-molecular-weight antibody immobilized onto the antibody immobilized region detected in the step (v), as a candidate for a light-chain variable region of the human antibody recognizing the specific antigen.

19. A method of producing a human antibody, the method comprising the step of producing the human antibody by combining the candidate for the light-chain variable region of the human antibody and the candidate for the heavy-chain variable region of the human antibody, the candidate for the light-chain variable region and the candidate for the heavy-chain variable region being determined by the method of screening as set forth in claim 15.

20. The antibody-immobilized carrier as set forth in claim 1, wherein:

the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody each being derived from an antibody recognizing a different antigen.

7. A method of producing an antibody-immobilized carrier comprising the step of:

immobilizing a heavy-chain low-molecular-weight antibody including a heavy-chain variable region and a light-chain low-molecular-weight antibody including a light-chain variable region separately onto a carrier, so that an antibody immobilized region is prepared.

8. The method as set forth in claim 7, wherein the step of immobilizing is repeated at least two times so that two or more of the antibody immobilized region are provided in an independent manner.

9. The method as set forth in claim 7, wherein the step of immobilizing comprising:

(a) the first sub-step of immobilizing, onto the carrier, insoluble aggregates of the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody by putting the denatured aggregates in contact with a carrier surface, the denatured insoluble aggregates each having been denatured by a denaturing agent and being in a denatured state; and

(b) the second sub-step of refolding the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody each in the denatured state, by removing the denaturing agent from the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody that are in the denatured state and immobilized.

10. The method as set forth in claim 9, wherein the first sub-step and the second sub-step in the step of immobilizing are carried out, separately for each of the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody.

11. The method as set forth in claim 10, wherein:

in the step of immobilizing, after the first sub-step and the second sub-step are carried out for the light-chain low-molecular-weight antibody, the first sub-step and the second sub-step are carried out for the heavy-chain low-molecular-weight antibody.

12. The method as set forth in claim 9, wherein:

the first sub-step employs, as the denaturing agent, urea whose concentration is in a range of 0.5 M to 4 M.

13. The antibody-immobilized carrier obtained by the method as set forth in claim 8.

21. The method as set forth in claim 7, wherein:

the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody each being derived from an antibody recognizing a different antigen.