High throughput generation and affinity maturation of humanized antibody

• US 9,464,286 B2
• Filed: 06/29/2006
• Issued: 10/11/2016
• Est. Priority Date: 08/12/2002
• Status: Active Grant

• Current Assignee: Adimab, LLC
• Original Assignee: Adimab, LLC
• Inventors: Zhu, Li, Wei, Shuanghong, Hua, Shaobing B.
• Primary Examiner(s): Goddard, Laura B
• Assistant Examiner(s): Natarajan, Meera
• Application Number:
  US11/480,037
• Publication Number:
  US 20060246515A1
• Time in Patent Office: 3,757 Days
• Field of Search: None
• 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 human or humanized antibodies in yeast, comprising:

assembling a library of antibodies in yeast cells, wherein the step of assembling the library comprises;

mutagenizing a V_H and V_L to produce a plurality of V_H and V_L; and

introducing the plurality of V_H and V_L into expression vectors by homologous recombination;

expressing the library of human or humanized antibodies in the yeast cells, wherein each of the human or humanized antibodies comprises a V_H and a V_L which form a V_H/V_L heterodimer in the yeast cells;

contacting a specific target protein with the library of human or humanized antibodies expressed in the yeast cells; and

selecting those human or humanized antibodies based on their binding to the target protein.

2. The method of claim 1, wherein a member of the library of humanized antibodies comprises:

V_H having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V_H grafted into a human antibody framework, and

V_L having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V_L grafted into a human antibody framework;

wherein

the one or more heavy chain CDR or CDRs and the one or more light chain CDR or CDRs are from the same non-human antibody, and the selected antibodies bind to the same target protein as the antibody from which the non-human CDRs are obtained.

3. The method of claim 1, wherein the library of humanized antibodies is generated by mutagenizing a chimeric antibody that comprises:

V_H having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V_H grafted into a human antibody framework, and

V_L having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V_L grafted into a human antibody framework, wherein

the one or more heavy chain CDR or CDRs and the one or more light chain CDR or CDRs are from the same non-human antibody, and the selected antibodies bind to the same target protein as the antibody from which the non-human CDRs are obtained.

4. The method of claim 1, wherein the V_H and the VL each is linked with a zipper domain by which the V_H and-the V_L are associated with each other to form a heterodimer.

5. The method of claim 4, wherein the zipper domains of the V_H and the V_L facilitate the heterodimerization of the V_H and the V_L through coiled-coil interactions.

6. The method of claim 4, wherein the zipper domains of the V_H and the V_L are leucine zippers.

7. The method of claim 4, wherein the zipper domains of the V_H and the V_L are leucine zippers formed by the leucine zippers from Fos and Jun.

8. The method of claim 4, wherein the zipper domains of the V_H and the V_L are leucine zippers formed by the leucine zippers from Myc and Max.

9. The method of claim 4, wherein the zipper domains of the V_H and the V_L each is linked to the C-terminus of the V_H and the V_L, respectively.

10. A method for screening a library of human or humanized antibodies in yeast, comprising:

assembling a library of antibodies in yeast cells, wherein the step of assembling the library comprises;

mutagenizing a V_H and V_L to produce a plurality of V_H and V_L; and

introducing the plurality of V_H and V_L into expression vectors by gap repair homologous recombination;

expressing the library of human or humanized antibodies in the yeast cells, wherein each of the human or humanized antibodies comprises a V_H and a V_L which form a V_H/V_L heterodimer in the yeast cells;

contacting a specific target protein with the library of human or humanized antibodies expressed in the yeast cells; and

selecting those human or humanized antibodies based on their binding to the target protein.

11. The method of claim 10, wherein a member of the library of humanized antibodies comprises:

V_H having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V_H grafted into a human antibody framework, and

V_L having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V_L grafted into a human antibody framework;

wherein

the one or more heavy chain CDR or CDRs and the one or more light chain CDR or CDRs are from the same non-human antibody, and the selected antibodies bind to the same target protein as the antibody from which the non-human CDRs are obtained.

12. The method of claim 10, wherein the library of humanized antibodies is generated by mutagenizing a chimeric antibody that comprises:

V_H having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V_H grafted into a human antibody framework, and

V_L having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V_L grafted into a human antibody framework, wherein

the one or more heavy chain CDR or CDRs and the one or more light chain CDR or CDRs are from the same non-human antibody, and the selected antibodies bind to the same target protein as the antibody from which the non-human CDRs are obtained.

13. The method of claim 10, wherein the V_H and the VL each is linked with a zipper domain by which the V_H and-the V_L are associated with each other to form a heterodimer.

14. The method of claim 13, wherein the zipper domains of the V_H and the V_L facilitate the heterodimerization of the V_H and the V_L through coiled-coil interactions.

15. The method of claim 13, wherein the zipper domains of the V_H and the V_L are leucine zippers.

16. The method of claim 13, wherein the zipper domains of the V_H and the V_L are leucine zippers formed by the leucine zippers from Fos and Jun.

17. The method of claim 13, wherein the zipper domains of the V_H and the V_L are leucine zippers formed by the leucine zippers from Myc and Max.

18. The method of claim 13, wherein the zipper domains of the V_H and the V_L each is linked to the C-terminus of the V_H and the V_L, respectively.