Directed evolution of novel binding proteins

• US 5,837,500 A
• Filed: 04/03/1995
• Issued: 11/17/1998
• Est. Priority Date: 09/02/1988
• Status: Expired due to Term

• Current Assignee: Dyax Corporation (Shire plc)
• Original Assignee: Dyax Corporation (Shire plc)
• Inventors: Ley, Arthur Charles, Kent, Rachel Baribault, Roberts, Bruce Lindsay, Gutterman, Sonia Kosow, Ladner, Robert Charles, Markland, William
• Primary Examiner(s): Ulm, John
• Application Number:
  US08/415,922
• Time in Patent Office: 1,324 Days
• Field of Search: 435/69.7, 435/172.3, 530/350, 530/412, 536/23.4
• US Class Current: 506/1
• 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 nuclecic acid encoding a proteinaceous binding domain that binds a predetermined target material comprising:

a) preparing a variegated population of filamentous phage, each phage including a nucleic acid construct coding for a chimeric potential binding protein, each said construct comprising DNA encoding (i) a potential binding domain which is a mutant of a predetermined parental binding domain, and (ii) an outer surface transport signs for obtaining the display of the potential binding domain on the outer surface of the phage, wherein said variegated population of phage collectively display a plurality of different potential binding domains, the differentiation among said plurality of different potential binding domains occuring through the at least partially random variation of one or more predetermined amino acid position of said parental binding domain to randomly obtain at each said position an amino belonging to a predetermined set of two or more amino acids, the amino acids of said set occuring at said position in predetermined expected proportions, said phage being separable on the basis of the potential binding domains displayed thereon;

b) causing the expression of said potential proteins and the display of said potential binding domains on the outer surface of said phage;

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

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 said phage are amplifiable in cell culture.

3. The method of claim 1 wherein, when the target is an antibody, the predetermined parental protein is not an antigen specifically bound by that antibody.

4. The method of claim 1 in which the parental binding doamain is an antibody domain.

5. A fusion protein consisting of a (a) amino terminal moiety which is a peptide or protein comprising an amino acid sequence foreign to a mature gIII protein of a filamentous phage, and (b) a carboxy terminal carrier protein moiety which comprises essentially all of said mature gIII protein, said fusion protein optionally including (c) a linker between said amino and carboxy terminal moieties, said carrier protein moiety acting, when thr fusion protein is produced in a suitable host cell infected by the phage, to cause the display of the fusion protein or a processed form thereof on the surafce of phage particles produced by the cell.

6. The fusion protein of claim 5 wherein the foreign peptide or protein is not an enzyme.

7. The fusion protein of claim 5 where the complex of the foreign peptide or protein with a target has a dissociation constant less than or equal to 10^-6 moles/liter.

8. The fusion protein of claim 5 wherein the carboxy terminal moiety corresponds to the entire mature gIII protein.

9. The fusion protein of claim 5 wherein the foreign peptide or protein is not found naturally in untransformed E. coli.

10. The fusion protein of claim 5 wherein the foreign peptide or protein is not found naturally in untransformed bacteria.

11. A virus bearing a fusion protein according to claim 5.

12. A library of virus according to claim 11, said library collectively displaying a plurality of different peptides or proteins.

13. The fusion protein of claim 5 in which the target is not an antibody.

18. The fusion protein of claim 11 in which the target is not an antibody.

14. A fusion protein consisting of (a) a foreign peptide or protein moiety comprising an amino acid sequence foreign to a mature gIII protein of a filamentous phage, and (b) a carrier protein moiety which is a functional portion of said mature gIII protein, said fusion protein optionally including (c) a linker foreign to said gIII protein between said amino and carboxy terminal moieties, said carrier protein corresponding to a carboxy portion of mature gIII protein obtainable by truncating the gIII protein at a domain boundary where a complex of the foreign peptide or protein with a target has a dissociation constant less than or equal to 10^-6 moles/liter.

15. The fusion protein of claim 14 wherein the foreign peptide or protein is not an enzyme.

16. The fusion protein of claim 14 wherein the polypeptide is obtainable by inserting the foreign peptide or protein at a domain boundary of the mature gIII protein.

17. The fusion protein of claim 14 wherein the carrier protein moiety corresponds to residue 275 through 424 of the M13 gIII protein.

19. A method of separating a predetermined target material from a predetermined contaminating material which comprises

(I) obtaining a nucleic acid encoding a binding protein having a proteinaceous binding domain that binds to said predetermined target material substantially more strongly than to said predetermined contaminating material, by a method comprising:

a) preparing a variegated population of amplifiable genetic packages, said genetic packages being selected from the group consisting of cells, spores and viruses, each said genetic package being genetically alterable and having an outer surface including a genetically determined outer surface protein, each package including a first nucleic acid construct coding for a chimeric potential binding protein, each said chimeric protein comprising and each said construct comprising DNA encoding (i) a potential binding domain which is a mutant of a predetermined domain of a predetermined parental protein other than a single chain antibody, and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of the genetic package, the expression of which construct results in the display of said chimeric potential binding protein and its potential binding domain on the outer surface of said genetic package; and

wherein said variegated population of genetic packages collectively display a plurality of different potential binding domains, the differentiation among said plurality of different potential binding domains occuring through the at least partially random variation of one or more predetermined amino acid positions of said parental binding domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occuring at said position in statistically predetermined expected proportions, said genetic packages being amplifiable in cell culture and separable on the basis of the potential binding domain displayed thereon,

(b) causing the expression of said chimeric potential binding proteins and the display of said potential binding domains on the outer surface of said packages;

(c) selecting for binding to the predetermined target material by

(i) contacting said package with the predetermined target material such that said potential binding domains and the target material may interact; and

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

(d) selecting for non-binding to the predetermined contaminating material by

(i) contacting said package with the predetermined contaminating material such that said potential binding domains and the contaminating material may interact;

(ii) separating packages dispalying a potential binding domain that binds the contaminating material from packages that do not so bind, and

(e) recovering at least one package displaying on its outer surface a chimeric binding protein comprising a successful binding domain (SBD) which bound said target material in step (c) and less strongly bound, if at all, said contaminating material in step (d), said package comprising nucleic acid encoding said successful binding domain, and amplifying said SBD-encoding nucleic acid in vivo or in vitro;

(II) producing a binding protein which is either said chimeric binding protein or a second binding protein having essentially the same binding domain;

(III) immobilizing or labeling said binding protein of (II), so as to form an affinity separation reagent; and

(IV) contacting a mixture comprising said target material and said contaminating material with said reagent such that said target material and said reagent can interact, and separating said target material from said contaminating material on the basis of their affinity for said affinity separation reagent.

20. A method of separating a predetermined target material from a predetermined contaminating material which comprises

(I) obtaining a nucleic acid encoding a binding protein having a proteinaceous binding domain that binds to said predetermined target material substantially more strongly than to said predetermined contaminating material, by a method comprising:

a) preparing a variegated population of phage, each phage including a first nucleic acid construct coding for a chimeric potential binding protein, each said chimeric protein comprising and each said construct comprising DNA encoding

(i) a potential binding domain which is a mutant of a predetermined domain of a predetermined parental protein, and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of the phage, the expression of which construct results in the display of said chimeric potential binding protein and its potential binding domain on the outer surface of said phage; and

wherein said variegated population of phage collectively display a plurality of different potential binding domains, the differentiation among said plurality of different potential binding domains occuring through the at least partially random variation of one or more predetermined amino acid position of said parental binding domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occuring at said position in statistically predetermined expected proportions, said phage being amplifiable by cultivation of phage-infected cells in cell culture and separable on the beats of the potential binding domain displayed thereon,

(b) causing the expression of said chimeric potential binding proteins and the display of said potential binding domains on the outer surface of said phage;

(c) selecting for binding to the predetermined target material by

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

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

(d) selecting for non-binding to the predetermined contaminating material by

(i) contacting said phage with the predetermined contaminating material such that said potential binding domains and the contaminating material may interact;

(ii) separating packages dispalying a potential binding domain that binds the contaminating 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 material in step (c) and less strongly bound said contaminating material in step (d), said phage comprising nucleic acid encoding said successful binding domain, and amplifying said SBD-encoding nucleic acid in vivo or in vitro;

(II) producing a binding protein which is either said chimeric binding protein or a second binding protein having essentially the same binding domain;

(III) immobilizing or labeling said binding protein of (II), so as to form an affinity separation reagent; and

(IV) contacting a mixture comprising said target material and said contaminating material with said reagent such that said target material and said reagent can interact, and separating said target material from said contaminating material on the basis of their affinity for said affinity separation reagent.

21. A mehtod of obtaining a nucleic acid encoding a binding protein haing a proteinaceous binding domain that binds a predetermined target material comprising:

a) preparing a variegated population of amplifiable genetic packages, said genetic packages being selected from the group consisting of cells, spores and viruses, each said genetic package being gentically alterable and having an outer surface including a genetically determined outer surface protein, each package including a first nucleic acid construct coding for a chimeric potential binding protein, each said chimeric protein comprising and each said construct comprising nucleic acid encoding (i) a potential binding domain which is a mutant of a predetermined domain of a predetermined parental protein other than a sigle chain antibdy, and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of the genetic package, the expression of which construct results in the display of said chimeric potential binding protein and its potential binding domain on the outer surface of said genetic package; and

wherein said varigated population of genetic packages collectively display a plurality of different potential binding domains, the differentiation among said plurality of different potential binding domains occuring through the at least partially random variation of one or more predetermined amino acid positions of said parental binding domain to randomly obtain at each said position an amino acid belonging to a predetermined set two or more amino acids, the amino acids of acid of said set occuring at said position in statistically predetermined expected proportions, said genetic packages being separable on the basis of the potential binding domain displayed thereon;

b) causing the expression of said chimeric potential binding proteins and the display of said potential binding domains on the outer surface of said packages;

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

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

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

22. The method of claim 21 in which said genetic packages are amplifiable in cell culture.

23. The method of claim 21 in which the SBD-encoding nucleic acid is amplified in vitro.

24. The method of claim 21 in which the genetic package is a replication-incompetent phage, and the isolated genetic package is rescued by helper phage and thereby amplified in vivo.

25. The method of claim 21 in which the genetic is package is a replication-incompetent phage, and the SBD-encoding nucleic acid is a,plified in vitro.

26. The method of claim 21 in which the parental binding domain is not an antibody domain.

27. The method of claim 21 where, when the target is an antibody, the predetermined parental protein is not an antigen specifically bound by that antibody,

with the further provise that at least one of the following conditions apply:

(1) in said step (a), the differentiation among said potential binding domains of said variegated population is limited to no more than about 20 predetermined amino acid resides of said sequence and where the premissible variation at said alterable reside positions is also predetermined,

(2) the genetic package is a single-stranded DNA bacteriophage other than bacteriophage lambda and the package is replicated in a bacterial host cell,

(3) the replicable genetic package is a bacterial cell and said DNA construct further comprises a periplasmic secretion signal sequence,

(4) the replicable genetic package is a bacterial spore,

(5) the potential binding proteins are intergeneric chimeric proteins,

(6) said population is characterized by the display of at least 10⁵ different potential binding domains,

(7) the population of replicable genetic packages of step (a) is obtained by;

i) preparing a variegated population of DNA inserts, said inserts comprising a plurality of variegated codons, which collectively encode a plurality of different potential binding domains, and

ii) incorporating the resulting population of DNA insets into the chosen replicable genetic packages to produce a variegated population of replicable genetic packages, and

(8) for any potentially encoded potential binding domain, the probability that if will be displayed by the at least one package in said population is at least 50%.

28. The method of claim 27 in which at least condition 1 applies.

29. The method of claim 27 in which at least condition 2 applies.

30. The method of claim 27 in which at least condition 3 applies.

31. The method of claim 27 in which at least condition 4 applies.

32. The method of claim 27 in which at least condition 5 applies.

33. The method of claim 27 in which at least condition 6 applies.

34. The method of claim 27 in which at least condition 7 applies.

35. The method of claim 27 in which at least condition 8 applies.

36. The method of claim 27 wherein the replicable genetic package is a filametous phage.

37. The method of claim 36 wherein the outer surface transport signal is provided by the major coat protein of a filamentous phage or a assemblable fragment thereof.

38. The method of claim 36 wherein the outer surface transport signal is provided by the gene III protein of a filamentous phage or an assemblable fragment thereof.

39. The method of claim 27 wherein the genetic package is a bacterial cell selected from the group consisting of strains of Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumonial, Neisseria gonorrhoeae, and Bacillus subtilis.

40. The method of claim 27 wherein the genetic package is a bacterial cell and the outer surface transport signal is derived from a bacterial outer surface protein selected from the group consisting of the lamB protein, OmpA, OmpC, OmpF, Phospholipase A, pilin, or an assemblable segment thereof.

41. The method of claim 40 wherein the chimeric surface protein substantially corresponds to LamB with the foreign potential binding domain inserted at 153.

42. The method of claim 21 in which the parental binding domain is an antibody domain.

43. The method of claim 21 in which the parental binding domain is not an antibody domain.