Molecular display system

Patent metadata

Field	Value
Publication number	US-10746743-B2
Application number	US-201615344971-A
Country	US
Kind code	B2
Filing date	Nov 7, 2016
Priority date	Sep 27, 2011
Publication date	Aug 18, 2020
Grant date	Aug 18, 2020

Abstract

There is provided herein a method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule by screening using molecular display in conjunction with the sequencing of positive and negative selection pools from the screen.

First claim

What is claimed is:

1. A method of identifying a potential binder to a cell surface antigen, the method comprising:
(a) providing a display library comprising clones, each of which displaying an antibody or antibody fragment, wherein the antibody or antibody fragment has an antibody variable region comprising:
CDR L1 comprising an amino acid sequence identified as SEQ ID NO: 240;
CDR L2 comprising an amino acid sequence identified as SEQ ID NO: 241;
CDR-L3 comprising an amino acid sequence identified as SEQ ID NO: 262;
CDR H1 comprising an amino acid sequence identified as SEQ ID NO: 263;
CDR H2 comprising an amino acid sequence identified as SEQ ID NO: 264; and
CDR-H3 comprising an amino acid sequence identified as SEQ ID NO: 265;
wherein each of the clones comprises DNA encoding the antibody variable region displayed thereby;
(b) screening the library against a population of cells that express the cell surface antigen to produce a positive selection pool of library clones and against a population of cells for producing a negative selection pool of library clones;
(c) sequencing DNAs encoding antibody variable regions of the positive selection pool and of the negative selection pool; and
(d) identifying, among the sequenced DNAs, DNA encoding an antibody variable region that is more abundant in the positive selection pool than in the negative selection pool,
thereby identifying an antibody variable region that is more abundant in the positive selection pool than in the negative selection pool, thereby identifying a potential binder to the cell surface antigen.

2. The method of claim 1 , further comprising a step of producing a purified antibody or antibody fragment, wherein the purified antibody or antibody fragment comprises the antibody variable region encoded by the DNA identified to be more abundant in the positive selection pool than in the negative selection pool.

3. The method of claim 1 , wherein the display library is a phage-display library and the clones are phage clones.

4. The method of claim 1 , wherein the antibody or antibody fragment is a Fab.

5. The method of claim 1 , wherein the cell surface antigen is a protein, wherein the cells that express the cell surface antigen are cells of a cell line which are transfected to express the cell surface antigen, and wherein the cells for producing the negative selection pool are cells of the cell line which are not transfected to express the cell surface antigen.

6. The method of claim 1 , wherein the sequencing is done by deep sequencing.

7. The method of claim 1 , wherein step (b) comprises multiple rounds of screening the library against the population of cells that express the cell surface antigen and against the population of cells for producing a negative selection pool of library clones.

8. The method of claim 1 , wherein step (d) comprises identifying DNA encoding an antibody variable region that is more abundant in the positive selection pool than in the negative selection pool by a factor selected from the group consisting of a factor of at least 2, a factor of at least 3, a factor of at least 4 and a factor of at least 5.

9. The method of claim 1 , wherein the cell surface antigen is selected from the group consisting of HER2, CD133, ErbB3, Fzd7, RORI, ROR2, exon16 deleted ErbB2, ITGA11 and O-GlcNAc.

10. A method of identifying a binder to a cell surface antigen, the method comprising:
(a) providing a display library comprising clones, each of which displaying an antibody or antibody fragment, wherein the antibody or antibody fragment has an antibody variable region comprising:
CDR L1 comprising an amino acid sequence identified as SEQ ID NO: 240;
CDR L2 comprising an amino acid sequence identified as SEQ ID NO: 241;
CDR-L3 comprising an amino acid sequence identified as SEQ ID NO: 262;
CDR H1 comprising an amino acid sequence identified as SEQ ID NO: 263;
CDR H2 comprising an amino acid sequence identified as SEQ ID NO: 264; and
CDR-H3 comprising an amino acid sequence identified as SEQ ID NO: 265;
wherein each of the clones comprises DNA encoding the antibody variable region displayed thereby;
(b) screening the library against a population of cells that express the cell surface antigen to produce a positive selection pool of library clones and against a population of cells for producing a negative selection pool of library clones;
(c) sequencing DNAs encoding antibody variable regions of the positive selection pool and of the negative selection pool;
(d) identifying, among the sequenced DNAs, DNA encoding an antibody variable region that is more abundant in the positive selection pool than in the negative selection pool; and
(e) producing a purified antibody or antibody fragment, wherein the purified antibody or antibody fragment comprises the antibody variable region encoded by the DNA identified to be more abundant in the positive selection pool than in the negative selection pool, and confirming that the purified antibody or antibody fragment binds to the cell surface antigen in purified form and/or binds to cells expressing the cell surface antigen,
thereby identifying a binder to the cell surface antigen.

11. The method of claim 10 , wherein the display library is a phage-display library and the clones are phage clones.

12. The method of claim 10 , wherein the antibody or antibody fragment is a Fab.

13. The method of claim 10 , wherein the cell surface antigen is a protein, wherein the cells that express the cell surface antigen are cells of a cell line which are transfected to express the cell surface antigen, and wherein the cells for producing the negative selection pool are cells of the cell line which are not transfected to express the cell surface antigen.

14. The method of claim 10 , wherein the sequencing is done by deep sequencing.

15. The method of claim 10 , wherein step (b) comprises multiple rounds of screening the library against the population of cells that express the cell surface antigen and against the population of cells for producing a negative selection pool of library clones.

16. The method of claim 10 , wherein step (d) comprises identifying DNA encoding an antibody variable region that is more abundant in the positive selection pool than in the negative selection pool by a factor selected from the group consisting of a factor of at least 2, a factor of at least 3, a factor of at least 4 and a factor of at least 5.

17. The method of claim 10 , wherein the cell surface antigen is selected from the group consisting of HER2, CD133, ErbB3, Fzd7, RORI, ROR2, exon16 deleted ErbB2, ITGA11 and O-GlcNAc.

18. The method of claim 1 , wherein the CDR-L3 comprises an isoleucine residue flanking the C-terminal amino acid residue thereof.

19. The method of claim 10 , wherein the CDR-L3 comprises an isoleucine residue flanking the C-terminal amino acid residue thereof.

Assignees

• Governing Council Univ Toronto
• Governing Council Univ Toronto

Inventors

• Sidhu Sachdev
• Moffat Jason
• Persson Helena
• Patel Nish
• Sundararajan Saravanan
• Gakhal Amandeep
• Ye Wei
• Tomic Jelena
• Mclaughlin Megan Elizabeth

Classifications

• G01N33/6854 Primary

  Immunoglobulins · CPC title

• C40B30/04 Primary

  by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding · CPC title

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• G01N33/6857

  Antibody fragments · CPC title