PROTEASE-RESISTANT SYSTEMS FOR POLYPEPTIDE DISPLAY AND METHODS OF MAKING AND USING THEREOF

• US 20160264961A1
• Filed: 03/28/2016
• Published: 09/15/2016
• Est. Priority Date: 08/10/2012
• Status: Active Grant

• Current Assignee: CytomX Therapeutics, Inc
• Original Assignee: CytomX Therapeutics, Inc
• Inventors: La Porte, Sherry Lynn, Moore, Stephen James, West, James William
• Granted Patent: US 10,947,531 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/21 containing a His-tag
• 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 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C40B 30/06 by measuring effects on living organisms, tissues or cells
• 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: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention generally relates to bacterial polypeptide display systems, libraries using these bacterial display systems, and methods of making and using these systems, including methods for improved display of polypeptides on the extracellular surface of bacteria using circularly permuted transmembrane bacterial polypeptides that have been modified to increase resistance to protease degradation and to enhance polypeptide display characteristics.

Claims

1. A composition comprising:

a carrier polypeptide (CP) comprising at least a transmembrane polypeptide portion, wherein the CP comprises an amino acid sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

56 and SEQ ID NO;

57, and

a displayed moiety (DM)

wherein the CP and the DM are operably linked.

2. The composition of claim 1, wherein the DM is displayed on the N-terminus of the CP.

3. The composition of claim 1, wherein the DM is displayed on the C-terminus of the CP.

4. The composition of claim 1, wherein the CP is a barrel-shaped beta sheet transmembrane polypeptide having a protease resistant sequence in at least one extracellular region of the polypeptide.

5. The composition of claim 4, wherein the CP comprises a protease-resistant sequence at the N-terminus and a protease resistant sequence at the C-terminus.

6. The composition of claim 4, wherein the CP comprises a protease resistant sequence in at least one extracellular loop of the polypeptide.

7. The composition of claim 4, wherein the protease resistant sequence is a glycine-serine rich peptide sequence.

8. The composition of claim 1, wherein the CP comprises the amino acid sequence of SEQ ID NO:

4 or SEQ ID NO;

58.

9. The composition of claim 1, wherein the CP is operably linked to the DM to form a display platform (DP) having the structural arrangement from N-terminus to C-terminus:

CP-DM or DM-CP.

10. The composition of claim 9, wherein the DP comprises a N-terminus tag, a C-terminus tag or both an N-terminus tag and a C-terminus tag.

11. The composition of claim 10, wherein the DP has the structural arrangement from N-terminus to C-terminus:

T1-CP-DM-T2 or T1-DM-CP-T2

12. The composition of claim 10, wherein the N-terminal tag comprises a protease-resistant sequence, the C-terminal tag comprises a protease-resistant sequence or both the N-terminal tag and the C-terminal tag comprises a protease-resistant sequence.

13. The composition of claim 10, wherein N-terminal tag comprises the amino acid sequence EYMPME (SEQ ID NO:

8).

14. The composition of claim 10, wherein the C-terminal tag comprises a histidine tag.

15. The composition of claim 14, wherein the histidine tag is an 8-His tag.

16. The composition of claim 10, wherein the N-terminal tag comprises the amino acid sequence EYMPME (SEQ ID NO:

8) and the C-terminal tag comprises an 8-His tag.

17. The composition of claim 10, wherein the DP comprises more than one DM.

18. The composition of claim 17, wherein the DP has the structural arrangement from N-terminus to C-terminus:

T1-DM1-DM2-CP-T2, T1-CP-DM1-DM2-T2, T1-DM1-DM2-DM3-CP-T2, T1-CP-DM1-DM2-DM3-T2, DM1-T1-DM2-CP-T2, T1-CP-DM1-T2-DM2, or T1-DM1-CP-DM2-T2.

19. The composition of claim 1, wherein the composition comprises one or more linkers.

20. The composition of claim 8, wherein the DP comprises one or more linkers.

21. The composition of claim 1, wherein the DM is selected from the group consisting of a substrate, an exosite, a masking moiety, an antibody, a receptor, a ligand, and other proteins, and any combinations thereof.

22. The composition of claim 21, wherein the substrate is a substrate for a protease.

23. An expression vector comprising a nucleic acid sequence encoding a composition of claim 1 operably linked to a plasmid.

24. A carrier protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

56 and SEQ ID NO;

57.

25. A composition comprising:

a replicable biological entity; and

a displayed moiety (DM) displayed on the surface of the replicable biological entity, wherein the DM is fused to an extracellular terminus of a circularly permuted bacterial outer membrane carrier protein (CP) to produce a display platform (DP), wherein the CP comprises an amino acid sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

56 and SEQ ID NO;

57.

26. The composition of claim 25, wherein the DM is a substrate for an enzyme.

27. The composition of claim 25, wherein the DP comprises a tag.

28. The composition of claim 27, wherein the tag is a fluorescent agent.

29. The composition of claim 27, wherein the tag is a C-terminal tag.

30. The composition of claim 27, wherein the tag is an N-terminal tag.

31. The composition of claim 25, wherein the C-terminus of the CP is exposed on the surface of the replicable biological entity.

32. The composition of claim 25, wherein the N-terminus of the CP is exposed on the surface of the replicable biological entity.

33. The composition of claim 25, wherein the replicable biological entity is a bacterial cell.

34. A polypeptide display library comprising:

a plurality of replicable biological entities, each member of the plurality comprising a displayed moiety (DM) displayed on the surface of the member replicable biological entity, wherein the DM is fused to an extracellular terminus of a circularly permuted bacterial outer membrane carrier protein (CP) to produce a display platform (DP), wherein the CP comprises an amino acid sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

56 and SEQ ID NO;

57.

35. The polypeptide display library of claim 34, wherein the DP comprises a tag.

36. The polypeptide display library of claim 35, wherein the tag is a fluorescent agent.

37. The polypeptide display library of claim 35, wherein the tag is a C-terminal tag.

38. The polypeptide display library of claim 35, wherein the tag is an N-terminal tag.

39. The polypeptide display library of claim 34, wherein the replicable biological entity is a bacterial cell.

40. A method for identifying a peptide substrate for an enzyme, comprising:

contacting a cell or other replicable biological entity with an enzyme, the cell comprising a cell outer membrane and expressing a peptide display scaffold, the peptide display scaffold comprising a fusion protein comprising a formula selected from the group consisting of;

T1-DM-carrier protein-T2 and 

 

(1)

T1-carrier protein-DM-T2 

 

(2)

wherein the carrier protein (CP) is a circularly permuted bacterial outer membrane protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

56 and SEQ ID NO;

57;

wherein DM comprises a member of a library of candidate peptide substrates for the enzyme;

wherein T1 and T2 are first and second detectable moieties; and

wherein T1-DM and T2 in formula (1) or T1 and DM-T2 in formula (2) are accessible at the extracellular surface of the cell outer membrane and the N- or C-terminus of the fusion protein is accessible at the extracellular surface of the cell outer membrane; and

wherein prior to contacting the cell with the enzyme, the cell exhibits a detectable T1 signal; and

detecting the presence of a detectable T2 signal in the absence of a detectable T1 signal, identifies the member of the library of candidate peptide substrates as a peptide substrate for the enzyme.

41. The method of claim 40, wherein the enzyme is a protease.

42. The method of claim 40, wherein said detecting is by fluorescence activated cell sorting.

43. The method of claim 40, wherein prior to contacting the cell with the enzyme, the cell exhibits a detectable T1 signal and a detectable T2 signal.

44. The method of claim 40, wherein T1 and T2 are not the same.

45. The method of claim 40, wherein the fusion protein further comprises at least one linker, wherein the linker is between DM and CP, the linker is between T2 and CP, the linker is between T1 and DM, or a combination thereof.

46. The method of claim 40, wherein detectable moieties T1 and T2 are affinity tags.

47. The method of claim 40, wherein T1-DM is located at an N-terminal domain of said fusion protein and T2 is located at a C-terminal domain of said fusion protein.

48. The method of claim 40, wherein T1-DM is located at a C-terminal domain of said fusion protein and T2 is located at an N-terminal domain of said fusion protein.

49. A peptide display scaffold, comprising a fusion protein comprising a formula selected from the group consisting of:

[T1-DM]-carrier protein-[T2] and 

 

(1)

[T1]-carrier protein-[DM-1-T2] 

 

(2)

wherein

the carrier protein is a circularly permuted bacterial outer membrane protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

56 and SEQ ID NO;

57;

DM comprises a candidate peptide; and

T1 and T2 are first and second detectable moieties;

and wherein, when the fusion protein is expressed in a cell comprising a cell outer membrane, T1-DM and T2 in formula (1) or T1 and DM-T2 in formula (2) are exposed at an extracellular surface of the cell outer membrane and one or both of the N- and C-termini of the fusion protein are exposed at the extracellular surface of the cell outer membrane.