Antibody and antibody mimetic for visualization and ablation of endogenous proteins

Patent metadata

Field	Value
Publication number	US-9746475-B2
Application number	US-201214004879-A
Country	US
Kind code	B2
Filing date	Mar 13, 2012
Priority date	Mar 14, 2011
Publication date	Aug 29, 2017
Grant date	Aug 29, 2017

Abstract

Provided are compositions and methods for labeling an endogenous protein, in particular, in a live cell, or for ablating an endogenous or target protein. The compositions relate to a fusion protein having a binding moiety such as an antibody, an antigen binding fragment of an antibody or an antibody mimetic that recognizes the endogenous or target protein.

First claim

What is claimed is:

1. A polynucleotide comprising:
(1) a nucleic acid sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises
(a) a binding moiety for recognizing an endogenous protein, and
(b) a transcription factor; and

(2) a transcription regulatory element operatively linked to the nucleic acid sequence, wherein the transcription regulatory element regulates the transcription of the binding moiety and the transcription factor regulates the activity of the transcription regulatory element.

2. A system for the expression of two or more polynucleotides, the system comprising:
(1) the polynucleotide of claim 1 ; and
(2) a second nucleic acid sequence encoding a second fusion polypeptide comprising
(a) a minimal promoter;
(b) a transcription factor that mediates expression of the binding moiety; and
(c) a heterologous protein; wherein the transcription factor regulates the minimal promoter causing expression of the heterologous protein.

3. The polynucleotide of claim 1 , wherein the binding moiety is one or more of an antibody mimetic, an intrabody, an antigen binding fragment of an antibody, a natural ligand, a monobody, a linear peptide, a lipocalin scaffold, an affibody scaffold or a DARPin scaffold.

4. The polynucleotide of claim 3 , wherein the intrabody comprises a fibronectin peptide.

5. The polynucleotide of claim 4 , wherein the fibronectin peptide comprises a 10FnIII fragment.

6. The polynucleotide of claim 1 , wherein the transcription factor comprises a DNA binding domain.

7. The polynucleotide of claim 6 , wherein the transcription factor is Gal4, LexA or a zinc finger domain.

8. The polynucleotide of claim 7 , wherein the transcription factor further comprises one or more of a regulatory domain, a transcriptional activator or an inducible promoter.

9. The polynucleotide of claim 7 , wherein the transcription factor comprises a DNA binding domain of Gal4 and a regulatory domain of Krab(A).

10. The polynucleotide of claim 1 , wherein the fusion polypeptide further comprises a reporter.

11. The polynucleotide of claim 10 , wherein the reporter is a green fluorescent protein (GFP), a blue fluorescent protein (BFP), a cyan fluorescent protein (CFP), a yellow fluorescent protein (YFP), mCherry, dTomato, mPlum, mOrange, mCitrine, Ypet, Cerulean CFP, luciferase, or β-galactosidase.

12. The polynucleotide of claim 1 , wherein the endogenous protein is a transmembrane protein, a nucleic protein, a cytoplasmic protein, a secreted protein, or an organelle protein.

13. The polynucleotide of claim 1 , wherein the endogenous protein is a transmembrane protein.

14. A DNA construct comprising the polynucleotide of claim 1 and a vector.

15. A composition comprising the polynucleotide of claim 1 and a carrier.

16. An isolated cell comprising the polynucleotide of claim 1 .

17. A method for labeling an endogenous protein in a cell, comprising contacting the cell with the polynucleotide of claim 10 , wherein the endogenous protein is labeled with the reporter.

18. The polynucleotide of claim 1 , wherein the fusion polypeptide further comprises a protein degradation signal.

19. A system comprising:
(1) the polynucleotide of claim 18 and
(2) a second nucleic acid sequence encoding a second fusion polypeptide comprising
(a) a minimal promoter;
(b) a transcription factor that mediates expression of the binding moiety; and
(c) a heterologous protein; wherein the transcription factor regulates the minimal promoter causing expression of the heterologous protein.

20. The polynucleotide of claim 18 , wherein the binding moiety comprises one or more of an antibody mimetic, an intrabody, an antigen binding fragment of an antibody, a natural ligand, a monobody, a linear peptide, a lipocalin scaffold, an affibody scaffold or a DARPin scaffold.

21. The polynucleotide of claim 20 , wherein the intrabody comprises a fibronectin peptide.

22. The polynucleotide of claim 18 , wherein the protein degradation signal induces protein degradation of a polypeptide encoded by the polynucleotide.

23. The polynucleotide or system of claim 22 , wherein the protein degradation is through ubiquitination, lysosomal degradation, or autophagy.

24. The polynucleotide or system of claim 23 , wherein the protein degradation is through ubiquitination.

25. The polynucleotide of claim 18 , wherein the protein degradation signal is a ubiquitin ligase, a HECT domain of an E6 protein, C20-WW-HECT, a Ring domain of Der3/Hrd1, a B-box domain of a TRIM protein, a U-box domain, KFERQ having the sequence SEQ ID NO: 1, Arg12 or Atg8/LC3.

26. The polynucleotide of claim 25 , wherein the ubiquitin ligase comprises one or more of a Ring domain of a protein X-linked mammalian inhibitor of apoptosis (XIAP), MDM2 or HDM2.

27. A DNA construct comprising the polynucleotide of claim 18 and a vector.

28. A composition comprising the polynucleotide of claim 18 and a carrier.

29. An isolated cell comprising the polynucleotide of claim 18 .

30. A method for inhibiting an activity of or degrading an endogenous protein in a cell, comprising contacting the cell with the polynucleotide of claim 18 , wherein the protein degradation signal causes the activity of the endogenous protein to be inhibited or degradation of the endogenous protein.

31. A composition comprising the polynucleotide of claim 10 and a second polynucleotide encoding a fusion polypeptide comprising a target peptide sequence and a cell localization domain, wherein the binding moiety recognizes the target peptide sequence.

32. A kit comprising the polynucleotide of claim 10 and a second polynucleotide encoding a fusion polypeptide comprising a target peptide sequence and a cell localization domain, wherein the binding moiety recognizes the target peptide sequence.

33. The composition of claim 31 , wherein the binding moiety is one or more of an antibody mimetic, an intrabody, an antigen binding fragment of an antibody, a natural ligand, a monobody, a linear peptide, a lipocalin scaffold, an affibody scaffold or a DARPin scaffold.

34. The composition of claim 33 , wherein the intrabody comprises a fibronectin peptide.

35. The composition of claim 34 , wherein the fibronectin peptide comprises a 10FnIII fragment.

36. The composition of claim 31 , wherein the cell localization domain localizes the chimeric polypeptide at Golgi apparatus, endoplasmic reticulum, lysosome, mitochondria, plasma membrane, or apical or basolateral domain of an epithelial cell.

37. The composition of claim 31 , wherein the reporter comprises a green fluorescent protein (GFP), a blue fluorescent protein (BFP), a cyan fluorescent protein (CFP), a yellow fluorescent protein (YFP), mCherry, dTomato, mPlum, mOrange, mCitrine, Ypet, Cerulean CFP, luciferase, or β-galactosidase.

Assignees

• Arnold Don B
• Roberts Richard W
• Gross Garrett G
• Mora Rudy
• Junge Jason
• Univ Southern California

Inventors

• Arnold Don B
• Roberts Richard W
• Gross Garrett G
• Mora Rudy
• Junge Jason

Classifications

• C07K16/18

  against material from animals or humans · CPC title

• G01N33/581 Primary

  with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates) · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• C07K14/78 Primary

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title