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

• US 8,901,045 B2
• Filed: 05/04/2012
• Issued: 12/02/2014
• Est. Priority Date: 04/17/2000
• Status: Expired due to Fees

• Current Assignee: Dyax Corporation (Shire plc)
• Original Assignee: Dyax Corporation (Shire plc)
• Inventors: Ladner, Robert C., Cohen, Edward H., Nastri, Horacio G., Rookey, Kristin L., Hoet, Rene, Mattheus Hoogenboom, Hendricus Renerus Jacobus
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US13/464,047
• Publication Number:
  US 20130040861A1
• Time in Patent Office: 942 Days
• Field of Search: None
• US Class Current: 506/17
• 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

Methods useful in constructing libraries that collectively display and/or express members of diverse families of peptides, polypeptides or proteins and the libraries produced using those methods are disclose. Methods of screening those libraries and the peptides, polypeptides or proteins identified by such screens are also disclosed.

Claims

1. A vector comprising:

(i) a DNA sequence encoding an antibody variable region linked to a fragment of a wild-type PIII anchor which does not mediate infection of phage particles; and

(ii) wild-type gene III;

wherein the nucleic acid sequence encoding the fragment of the wild-type pIII anchor differs from the wild-type gene III to an extent sufficient to reduce homologous recombination between the nucleic acid encoding the fragment of the wild-type PIII anchor and the wild-type gene III,

wherein all the codons in the nucleic acid encoding the fragment of the wild-type pIII anchor have at least one base change introduced per codon compared to the wild-type gene III.

2. The vector of claim 1, wherein the DNA encodes a Fab.

3. The vector of claim 1, wherein the DNA encodes heavy chain VHCH1.

4. The vector of claim 3, wherein the heavy chain VHCH1 is linked to trpIII.

5. The vector of claim 1, wherein the DNA encodes light chain VLCL.

6. The vector of claim 5, wherein the light chain VLCL is linked to trpIII.

7. The vector of claim 1, wherein the DNA encodes scFv.

8. The vector of claim 7, wherein the scFv is VL-VH.

9. The vector of claim 7, wherein the scFv is VH-VL.

10. The vector of claim 1, wherein the DNA sequence encoding the antibody variable region further comprises an inducible promoter.

11. The vector of claim 10, wherein the inducible promoter regulates expression of the DNA sequence encoding the antibody variable region.

12. The vector of claim 1, wherein the DNA sequence encoding the antibody variable region further comprises an amber stop codon.

13. The vector of claim 12, wherein the amber stop codon is located between the antibody variable region and the fragment of the wild-type PIII anchor.

14. The vector of claim 1, wherein the vector is phage or phagemid.