Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries

• US 8,288,322 B2
• Filed: 10/25/2001
• Issued: 10/16/2012
• Est. Priority Date: 04/17/2000
• Status: Expired due to Term

• Current Assignee: Takeda Pharmaceutical Company Limited
• Original Assignee: Dyax Corporation (Shire plc)
• Inventors: Ladner, Robert C., Cohen, Edward H., Nastri, Horacio G., Rookey, Kristin L., Hoet, Rene, Hoogenboom, Hendricus Renerus Jacobus Mattheus
• Primary Examiner(s): Marschel, Ardin
• Assistant Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US10/045,674
• Publication Number:
  US 20030232333A1
• Time in Patent Office: 4,009 Days
• Field of Search: None
• US Class Current: 506/18
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• 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
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

A library containing a collection of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and that collectively display at least a portion of the family, the displayed peptides, polypeptides or proteins being encoded by DNA sequences containing sequences encoding a heavy chain CDR region is described.

Claims

1. A library comprising a collection of members of a family, the family comprising a diversity of antibodies or functional fragments thereof, wherein the antibodies or functional fragments thereof each comprise a VH CDR1, a VH CDR2, a VH CDR3 and framework regions and the antibodies or functional fragments are encoded by DNA sequences comprising sequences encoding

(a) the VH CDR1, wherein the VH CDR1 comprises the amino acid sequence according to the formula -X₁-Y-X₂-M-X₃- (SEQ ID NO:

636), wherein X₁, X₂, and X₃ are independently selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y,

(b) the VH CDR2, wherein the VH CDR2 comprises the amino acid sequence according to the formula X₄-I-X₅-X₆-S-G-G-X₇-T-X₈-Y-A-D-S-V-K-G- (SEQ ID NO;

637), wherein X₄ and X₅ are independently selected from the group consisting of Y, R, W, V, G, and S, X₆ is selected from the group consisting of P and S, and X₇ and X₈ are independently selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y,

(c) the VH CDR3, wherein the sequence encoding the VH CDR3 is from the CDR3 region of an immunoglobulin gene of a B cell, and

(d) the framework regions, wherein the framework regions comprises VH 3-23 framework regions or VH3-23 related germline framework regions.

2. The library according to claim 1, wherein the antibodies or functional fragments thereof further comprise an immunoglobulin light chain, and said DNA sequences further comprise a sequence encoding the immunoglobin light chain.

3. The library according to claim 2, wherein said sequence encoding an immunoglobin light chain is from a B cell.

4. The library according to claim 3, wherein said B cell is from a blood sample from an autoimmune patient.

5. The library according to claim 4, wherein the autoimmune patient is diagnosed with a disorder selected from the group consisting of systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphospholipid syndrome and vasculitis.

6. The library according to claim 1, wherein said antibodies or functional fragments thereof are linked via a short linker to the final portion of M13 gene III.

7. The library according to claim 1, wherein the diversity of antibodies or functional fragments thereof is displayed on genetic packages.

8. The library according to claim 7, wherein said genetic packages are M13 phage.

9. The library according to claim 8, wherein said DNA sequences are in a phage vector.

10. The library according to claim 9, wherein said phage vector comprises a wild-type gene iii and a truncated gene iii for display of the antibody or functional fragment thereof.

11. The library according to claim 8, wherein said DNA sequences are in a phagemid vector.

12. The library according to claim 8, wherein said displayed antibodies or functional fragments thereof are linked via a short linker to the final portion of M13 gene III.

13. The library according to claim 2, wherein the diversity of antibodies or functional fragments thereof is displayed on genetic packages.

14. The library according to claim 13, wherein said genetic packages are M13 phage.

15. The library according to claim 14, wherein said DNA sequences are in a phage vector.

16. The library according to claim 14, wherein said DNA sequences are in a phagemid vector.

17. The library according to claim 14, wherein said displayed antibodies or functional fragments thereof are linked via a short linker to the final portion of M13 gene III.

18. The library of claim 1, wherein the functional fragments are Fab fragments or scFv fragments.