High throughput generation and affinity maturation of humanized antibody

• US 20040067532A1
• Filed: 06/11/2003
• Published: 04/08/2004
• Est. Priority Date: 08/12/2002
• Status: Abandoned Application

• Current Assignee: Adimab, LLC
• Original Assignee: Genetastix Corp.
• Inventors: Zhu, Li, Hua, Shaobing B., Wei, Shuanghong
• Application Number:
  US10/460,595
• Publication Number:
  US 20040067532A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1041 Ribosome/Polysome display, e.g. SPERT, ARM
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

Compositions, methods, and kits are provided for efficiently generating and screening humanized antibody with high affinity against a specific antigen. The library of humanized antibody is generated by mutagenizing a chimeric antibody template that combines human antibody framework and antigen binding sites of a non-human antibody. Alternatively, the library of humanized antibody is generated by grafting essential antigen-recognition segment(s) such as CDRs of the non-human antibody into the corresponding position(s) of each member of a human antibody library. This library of humanized antibody is then screened for high affinity binding toward a specific antigen in vivo in organism such as yeast or in vitro using techniques such as ribosome display or mRNA display. The overall process can be efficiently performed in a high throughput and automated manner, thus mimicking the natural process of antibody affinity maturation.

Claims

1. A method for screening a library of humanized antibody in yeast, comprising:

expressing a library of humanized antibodies in yeast cells;

expressing a specific target protein in the yeast cells expressing the humanized antibodies; and

selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by binding of the humanized antibody to the target protein.

2. The method of claim 1, wherein a member of the library of humanized antibody contains one or more CDRs of a non-human antibody grafted into a human antibody framework.

3. The method of claim 1, wherein the library of humanized antibody are generated by mutagenizing a chimeric antibody that contains one or more CDRs of a non-human antibody grafted into a human antibody framework.

4. A method for screening a library of humanized antibody in vitro, comprising:

expressing a library of humanized antibodies in vitro;

displaying the library of humanized antibodies on ribosome;

contacting the ribosome displaying the library of humanized antibodies with a target antigen; and

selecting those ribosomes that bind to the target antigen,

5. The method of claim 4, wherein the target antigen is immobilized to a substrate.

6. The method of claim 4, wherein the target antigen is fluorescence labeled.

7. A method for screening a library of humanized antibody in vitro, comprising:

transcribing a library of cDNA encoding a library of humanized antibodies in vitro to produce a library of mRNA;

contacting the library of mRNA with a peptidyl acceptor linker to produce a library of modified mRNA;

translating the library of modified mRNA in vitro to produce a library of humanized antibody, wherein the translated humanized antibody is covalently linked to the modified mRNA by the peptidyl acceptor linker; and

contacting the library of humanized antibodies linked with the modified mRNA with a target antigen; and

selecting the humanized antibody that bind to the target antigen,

8. The method of claim 7, wherein the peptidyl acceptor linker is puromycin.