Directed evolution of novel binding proteins

• US 6,979,538 B2
• Filed: 02/14/2001
• Issued: 12/27/2005
• Est. Priority Date: 09/02/1988
• Status: Expired due to Fees

• Current Assignee: Dyax Corporation (Shire plc)
• Original Assignee: Dyax Corporation (Shire plc)
• Inventors: Ladner, Robert Charles, Ley, Arthur Charles, Kent, Rachel Baribault, Markland, William, Roberts, Bruce Lindsay, Guterman, Sonia Kosow
• Primary Examiner(s): Wang, Andrew
• Application Number:
  US09/781,988
• Publication Number:
  US 20020150881A1
• Time in Patent Office: 1,777 Days
• Field of Search: 435/69.7, 435/7.32, 435/7.2, 435/7.1, 435/6, 435/5, 435/4, 435/DIG.5, 435/DIG.3, 435/DIG.2, 530/350, 536/23.4
• US Class Current: 435/6.16
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 1/107 by chemical modification of precursor peptides
• CPC Class Codes: C07K 14/43522 from scorpions
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C07K 14/8114 Kunitz type inhibitors
• CPC Class Codes: C07K 14/8117 Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
• 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 2795/14111 Inoviridae
• CPC Class Codes: C12N 2795/14122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2795/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• 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

In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

Claims

1. A method of obtaining a nucleic acid encoding a proteinaceous binding domain that binds a predetermined target material comprising:

(a) expressing a plurality of different chimeric potential binding proteins, each said protein comprising (i) a potential binding domain which is an antibody variable domain and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of a filamentous phase,

(b) displaying said chimeric potential binding proteins on the outer surfaces of a population of filamentous phage, said population thereby collectively displaying a plurality of different potential binding domains which differ from each other in amino acid sequence, each phage including a nucleic acid construct comprising a nucleic acid sequence encoding one of said binding domains;

(c) contacting said phage with the target material such that said potential binding domains and the target material may interact;

(d) separating phage displaying a potential binding domain that binds the target material from phage that do not so bind, and

(e) recovering at least one phage displaying on its outer surface a chimeric binding protein comprising a successful binding domain (SBD) which bound said target, said phage enclosing SBD-encoding nucleic acid, and amplifying said SBD-encoding nucleic acid in vivo or in vitro.

2. The method of claim 1 wherein, in said step (a), the differentiation among said potential binding domains is limited to no more than 20 amino acid residue positions of said domains.

3. The method of claim 1 in which each binding domain has one or more variable positions and one or more non-variable positions, the differences in amino acid sequence arising from the amino acid variation at said variable positions, and one or more variable positions correspond to positions in the hypervariable region of naturally occurring antibody variable domains.

4. The method of claim 1 in which said population of filamentous phage is obtained by subcloning a mixture of DNA encoding a plurality of different chimeric proteins, comprising different potential binding domains.

5. The method of claim 4 in which said mixture of DNA is synthesized, without a template, by nonbiological means.

6. The method of claim 5 in which in which the potential binding domains of said mixed population differ at positions predetermined prior to synthesis of said mixture of DNA.

7. The method of claim 1 in which the outer surface transport signal is an outer surface protein of said phage or a portion thereof functional to direct said display.

8. The method of claim 1 in which the phage is a class I filamentous phase.

9. The method of claim 8 in which the phage is an F pilus-specific phage.

10. The method of claim 1 in which the phage is an M13, fl, or fd phage.

11. The method of claim 1 in which the phage is a class II filamentous phage.

12. The method of claim 1 in which the phage is a Pfl, Xf, or Pf3 phage.

13. The method of claim 1 in which the successful binding domain has an affinity for the target material such that its dissociation constant is less than 10–

6 moles/liter.

14. The method of claim 1 in which the successful binding domain has an affinity for the target material such that its dissociation constant is less than 10″

⁷ moles/liter.

15. The method of claim 13 in which the phage is an M13, fl, or fd phage.

16. The method of claim 3 in which all of the variable positions correspond to positions in the hypervariable region of naturally occurring antibody variable domains.

17. The method of claim 1 in which the potential binding domains are each at least 88% identical to a naturally occurring antibody variable domain at amino acid positions which do not correspond to the hypervariable region of the naturally occurring antibody variable domain.

18. The method of claim 1 in which the melting point of the domain is at least 40°

C.

19. The method of claim 1 in which the melting point of the domain is at least 50°

C.