DESIGN AND GENERATION OF HUMAN DE NOVO pIX PHAGE DISPLAY LIBRARIES

• US 20100021477A1
• Filed: 08/25/2009
• Published: 01/28/2010
• Est. Priority Date: 12/19/2007
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Reddy, Ramachandra, Knight, David M., Shi, Lei, Tornetta, Mark, Chen, Qiang, Sweet, Raymond W., Wheeler, John, Whitaker, Brian, Amegadzie, Bernard, Almagro, Juan C., Borozdina-Birch, Lionella, Lu, Jin, Luo, Jinquan, Tsui, Ping
• Granted Patent: US 9,062,305 B2
• Time in Patent Office: Days
• US Class Current: 424/164.100
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• 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: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

Described and claimed herein are combinatorial synthetic Fab libraries displayed on a phage pIX protein. The libraries were built on scaffolds representing the most frequently used genes in human antibodies, which were diversified to mirror the variability of natural antibodies. After selection using a diverse panel of proteins, numerous specific and high-affinity Fabs were isolated. By a process called in-line maturation the affinity of some antibodies was improved up to one hundred-fold yielding low pM binders suitable for in vivo use. This work thus demonstrates the feasibility of displaying complex Fab libraries as pIX-fusion proteins for antibody discovery and lays the foundations for studies on the structure-function relationship of antibodies.

Claims

1. A recombinant nucleic acid phage vector comprising nucleic acid sequences encoding a phage pIX protein fused to a Fab amino acid sequence, wherein said Fab amino acid sequence binds to an antigen.

2. The phage vector of claim 1 wherein said Fab amino acid sequence is a sequence selected from the group consisting of an Fd sequence and a light chain sequence.

3. The phage vector of claim 1 further comprising:

a. a nucleic acid encoding a leader peptide;

b. a nucleic acid encoding a recombinant tag, promoter, or selectable marker;

c. a nucleic acid encoding a variable heavy chain sequence;

d. a nucleic acid encoding a portion of or a full length constant heavy chain;

e. a nucleic acid encoding a recombinant pIX protein;

f. a nucleic acid encoding a variable light chain that selectively binds to said antigen; and

g. a nucleic acid encoding a portion of or a full length constant light chain.

4. A nucleic acid phage vector according to claim 3, wherein said nucleic acid encoding said leader peptide is a pelB sequence.

5. A nucleic acid phage vector according to claim 3, wherein said promoter is an inducible promoter and is a lac promoter or mutant of lac.

6. A nucleic acid phage vector according to claim 3, wherein said antigen is part of a biologically active molecule that mediates at least one biological in vivo activity.

7. A bacterial host cell comprising a nucleic acid phage vector according to claim 1.

8. A biologically active fusion protein expressed by a bacterial host cell according to claim 7.

9. A biologically active Fab antibody fragment variant derived from said fusion protein according to claim 8.

10. A phage library of bacterial host cells comprising a nucleic acid phage vector according to claim 1.

11. The phage library of claim 10, wherein said nucleic acids encode a Fab variant.

12. The phage library of claim 11 wherein said Fab variant was constructed by combining germline variable light chain nucleic acid sequences with variable heavy chain nucleic acid sequences that comprise one or more mutations relative to germline variable heavy chain nucleic acid sequences.

13. The phage library of claim 11 wherein said Fab variant was constructed by combining variable light chain nucleic acid sequences with variable heavy chain nucleic acid sequences, wherein said sequences comprise one or more mutations relative to germline variable light chain and variable heavy chain nucleic acid sequences.

14. A phage library according to claim 11, wherein said Fab variant comprises a plurality of diverse variations in the germline heavy chain complementarity determining regions selected from the group consisting of CDR 1, CDR2, and CDR3.

15. A phage library according to claim 12, wherein said phage library is further comprised of Fab antibody fragments comprising sequence variants wherein said Fab antibody fragments have improved binding affinity relative to prior versions of an antibody that binds to a biologically active ligand.

16. A phage library according to claim 15, wherein said Fab antibody fragments are selected for increased affinity or biological activity.

17. A phage library according to claim 16, wherein said selection is conducted as part of the library generation process or in parallel using a subset of said phage library.

18. A method for screening a phage library for Fab antibody fragments having a desired biological activity, comprising (a) expressing Fab antibody fragments from a phage library according to claim 10, and (b) selecting bacterial cells expressing said Fab antibody fragments having said desired biological activity.

19. A Fab antibody fragment encoding nucleic acid, obtained from a method according to claim 16.

20. A pharmaceutical composition comprising a Fab antibody fragment selected using the method of claim 18.