HIGH THROUGHPUT GENERATION AND AFFINITY MATURATION OF HUMANIZED ANTIBODY

• US 20160237424A1
• Filed: 02/22/2016
• Published: 08/18/2016
• Est. Priority Date: 08/12/2002
• Status: Active Grant

• Current Assignee: Adimab, LLC
• Original Assignee: Adimab, LLC
• Inventors: Wei, Shuanghong, Zhu, Li, Hua, Shaobing B.
• Granted Patent: US 10,329,555 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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.