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Last Updated: March 28, 2024

Claims for Patent: 5,403,484


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Summary for Patent: 5,403,484
Title: Viruses expressing chimeric binding proteins
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.
Inventor(s): Ladner; Robert C. (Ijamsville, MD), Guterman; Sonia K. (Belmont, MA), Roberts; Bruce L. (Milford, MA), Markland; William (Milford, MA), Ley; Arthur C. (Newton, MA), Kent; Rachel B. (Boxborough, MA)
Assignee: Protein Engineering Corporation (Cambridge, MA)
Application Number:08/009,319
Patent Claims:1. A virus bearing on its outer surface a chimeric binding protein, said protein comprising (i) a proteinaceous binding domain, other than a single chain antibody, which is sufficiently stable in structure to have a melting point of at least 40.degree. C., and which binds to a target:, other than the variable domain of an antibody, sufficiently strongly so that the dissociation constant of the binding domain: target complex is less than 10.sup.-6 moles/liter, and (ii) at least a functional portion of a coat protein of said virus, said portion acting, when the chimeric protein is produced in a suitable host cell, to cause the display of the chimeric binding protein or a processed form thereof on the outer surface of the virus, said binding domain being capable of binding to a target material which said coat protein does not preferentially bind, said binding domain being foreign to the native coat proteins of said virus.

2. The virus of claim 1 wherein the virus is a phage.

3. The virus of claim 2 wherein the phage is a filamentous phage.

4. The virus of claim 3 wherein the coat protein is the gIII protein.

5. The virus of claim 3 wherein the coat protein is the gVIII protein.

6. The virus of claim 1 wherein the proteinaceous binding domain has a melting point of at least 50.degree. C.

7. The virus of claim 1 wherein the proteinaceous binding domain features at least two disulfides.

8. The virus of claim 1 wherein the proteinaceous binding domain has a single disulfide bond and the span of the bond is not more than nine amino acids.

9. The virus of claim 1 wherein the proteinaceous binding domain contains no more than 30 residues and at least 2 disulfides.

10. The virus of claim 1 wherein the proteinaceous binding domain contains no more than 60 residues and at least 3 disulfides.

11. The virus of claim 1 wherein the proteinaceous binding domain contains no more than 80 residues and at least 4 disulfides.

12. The virus of claim 1 wherein the proteinaceous binding domain is a protease inhibitor.

13. The virus of claim 12 wherein the proteinaceous binding domain is a serine protease inhibitor.

14. The virus of claim 1 wherein the proteinaceous binding domain is at least 50% homologous with BPTI.

15. The virus of claim 1 wherein the chimeric protein further comprises a linker peptide, linking said binding domain to said coat protein or functional portion thereof, which is specifically cleavable by a site-specific protease.

16. The virus of claim 15 wherein the site-specific protease is Factor Xa, Factor XIa, Kallikvein, thrombin, Factor XIIa, collagenase or enterokinase.

17. A library of virus according to claim 1, said library collectively displaying a plurality of different binding domains.

18. The library of claim 17 wherein at least 10.sup.4 different proteinaceous binding domains are displayed.

19. The library of claim 17, wherein at least some of the displayed binding domains have amino acid sequences which are not identical to an amino acid sequence encoded by a naturally occurring DNA sequence.

20. The virus of claim 1 wherein the proteinaceous binding domain has an amino acid sequence which is not identical to an amino acid sequence encoded by a naturally occurring DNA sequence.

21. The virus of claim 1 wherein the amino acid sequence differences among the binding domains of said library are limited to no more than about 20 corresponding amino acid residue positions of said domains.

22. The virus of claim 1 wherein the binding domain is at least substantially homologous with a binding domain selected from the group consisting of the binding domains of bovine pancreatic trypsin inhibitor, crambin, Cucurbita maxima trypsin inhibitor III, heat stable enterotoxin of Escherichia coli, .alpha. Conotoxin GI, .lambda. Conotroxin GIII, .omega. Conotoxin GIV, apamin, charybdtoxin, secretory leukocyte protease inhibitor, cystatin, eglin, barley protease inhibitor, ovomucoid, T4 lysozyme, hen egg white lysozyme, ribonuclease, azurin, tumor necrosis factor, and CD4.

23. The virus of claim 1, said virus further bearing on its outer surface the corresponding wild-type coat protein of said virus.

24. A library of virus according to claim 23.

25. The virus of claim 1 wherein the proteinaceous binding domain is coupled essentially to the amino terminal of the mature coat protein.

26. A chimeric binding protein comprising (i) a proteinaceous binding domain, other than a single chain antibody, which is sufficiently stable in structure to have a melting point of at least 40.degree. C., and which binds to a target, other than the variable domain of an antibody, sufficiently strongly so that the disassociation constant of the binding domain: target complex is less that 10.sup.-6 moles/liter, and (ii) at least a functional portion of a coat protein of a virus, said portion acting, when the chimeric protein is produced in a suitable host cell, to cause the display of the chimeric binding protein or a processed form thereof on the outer surface of the virus, said binding domain being capable of binding to a target material which said coat protein does not preferentially bind, said binding domain being foreign to the native coat proteins of said virus.

27. The protein of claim 26 wherein the virus is a phage.

28. The protein of claim 27 wherein the phage is a filamentous phage.

29. The protein of claim 28 wherein the coat protein is the gIII protein.

30. The protein of claim 28 wherein the coat protein is the gVIII protein.

31. The protein of claim 26 wherein the binding domain has a melting point of at least 50.degree. C.

32. The protein of claim 26 wherein the binding domain features at least two disulfides.

33. The protein of claim 26 wherein the proteinaceous binding domain has a single disulfide bond and the span of the bond is not more than nine amino acids.

34. The protein of claim 26 wherein the proteinaceous binding domain contains no more than 30 residues and at least 2 disulfides.

35. The protein of claim 26 wherein the proteinaceous binding domain contains no more than 60 residues and at least 3 disulfides.

36. The protein of claim 28 wherein the proteinaceous binding domain contains no more than 80 residues and at least 4 disulfides.

37. The protein of claim 26 wherein the proteinaceous binding domain is a protease inhibitor.

38. The protein of claim 37 wherein the proteinaceous binding domain is a serine protease inhibitor.

39. The protein of claim 26 wherein the proteinaceous binding domain is at least 50% homologous with BPTI.

40. The protein of claim 26 wherein the chimeric binding protein further comprises a linker peptide, linking said binding domain to said coat protein or functional portion thereof, which is specifically cleavable by a site-specific protease.

41. The protein of claim 40 wherein the site-specific protease is Factor Xa, Factor XIa, Kallikvein, thrombin, Factor XIIa, collagenase or enterokinase.

42. The protein of claim 26 wherein the proteinaceous binding domain has an amino acid sequence which is not identical to an amino acid sequence encoded by a naturally occurring DNA sequence.

43. The protein of claim 26 wherein the proteinaceous binding domain is coupled essentially to the amino terminal of the mature coat protein.

44. A fusion protein comprising (a) a carrier protein moiety essentially corresponding to a mature gene III protein of a filamentous phage, said carrier protein moiety acting, when the 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 surface of the phage, and (b) a foreign peptide or protein coupled to the amino terminal of said carrier protein moiety.

45. A recombinant filamentous phage bearing a fusion protein according to claim 44, upon its outer surface, said carrier protein moiety being integrated into the coat of the phage, said foreign peptide or protein being capable of binding specifically to a target which said phage does not specifically bind and being of an amino acid sequence foreign to the coat proteins native to said phage.

46. A library of recombinant phage according to claim 45, said library displaying a plurality of different foreign peptides or proteins.

47. A fusion protein comprising (a) at least a functional portion of a mature gene VIII protein of a filamentous phage, said portion acting, when the 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 surface of the phage, and (b) a foreign peptide or protein coupled to said functional portion of said mature gene VIII protein.

48. A recombinant filamentous phage bearing a fusion protein according to claim 47, upon its outer surface, said functional portion of the gene VIII protein being integrated into the coat of the phage, said foreign peptide or protein being capable of binding specifically to a target which said phage does not specifically bind and being of an amino acid sequence foreign to the coat proteins native to said phage.

49. A library of recombinant phage according to claim 48, said library displaying a plurality of different foreign peptides or proteins.

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