Fusion protein comprising targeting moiety, cleavage site, and cell membrane penetrating domain, and use thereof

Patent metadata

Field	Value
Publication number	US-9617344-B2
Application number	US-201514794542-A
Country	US
Kind code	B2
Filing date	Jul 8, 2015
Priority date	Jul 8, 2014
Publication date	Apr 11, 2017
Grant date	Apr 11, 2017

Abstract

A fusion protein including a targeting moiety, a cleavage site, and a cell membrane penetrating domain, a conjugate including the fusion protein and a bioactive molecule, and methods employing the fusion protein and the conjugate are provided.

First claim

What is claimed is:

1. A fusion protein comprising a targeting moiety, a cleavage site, and a cell membrane penetrating domain, wherein
the targeting moiety is an antibody or antigen-binding fragment thereof, a designed ankyrin repeat protein (DARPin), or a combination thereof, that specifically binds an ErbB family receptor tyrosine kinase protein;
the cleavage site is a recognition site of a matrix metalloproteinase (MMP), cathepsin, or urokinase-type plasminogen activator (uPA); and
the cell membrane penetrating domain is a membrane-translocation sequence, a macromolecule intracellular transduction domain, a fusion peptide comprising a hydrophobic peptide and a basic peptide, or a combination thereof.

2. The fusion protein of claim 1 , wherein the targeting moiety is an antibody or antigen-binding fragment thereof, a DARPin, or a combination thereof, that specifically binds epidermal growth factor receptor (EGFR).

3. The fusion protein of claim 2 , wherein the targeting moiety is at least one anti-EGFR DARPin selected from SEQ ID NOs: 7 to 10.

4. The fusion protein of claim 1 , wherein the cell membrane penetrating domain comprises SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 38, or a combination thereof.

5. The fusion protein of claim 1 , wherein the cell membrane penetrating domain is a fusion peptide comprising a hydrophobic peptide and a basic peptide, wherein
the hydrophobic peptide comprises a total of about 5 to about 40 amino acids of which 70% or more are hydrophobic amino acids, and each hydrophobic amino acid is independently selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, and phenylalanine, and
the basic peptide consists of a basic peptide unit consisting of about 1 to about 6 basic amino acids or a repeat comprising 2 to 6 basic peptide units.

6. The fusion protein of claim 5 , wherein the hydrophobic peptide comprises SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a combination thereof.

7. The fusion protein of claim 5 , wherein the basic peptide comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

8. The fusion protein of claim 5 , wherein the basic peptide is linked to the C-terminus of the hydrophobic peptide.

9. The fusion protein of claim 1 , comprising SEQ ID NO: 30.

10. A pharmaceutical composition comprising the fusion protein of claim 1 .

11. A conjugate comprising the fusion protein of claim 1 and a bioactive molecule, wherein the bioactive molecule is a protein, a peptide, a nucleic acid, a small-molecule drug, or a combination thereof.

12. The conjugate of claim 11 , wherein the bioactive molecule is a p16 protein variant comprising SEQ ID NO: 1 with an amino acid substitution at one or more of positions 15 (W15), 37 (L37), 65 (L65), 72 (C72), 78 (L78), 106 (V106), and 113 (L113) independently with lysine (K), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), serine (S), alanine (A), threonine (T), or arginine (R).

13. The conjugate of claim 12 , wherein the amino acid substitution is at least one selected from the group consisting of:
a substitution of tryptophan at position 15 (W15) with lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), or serine (S),
a substitution of leucine at position 37 (L37) with aspartic acid (D), arginine (R), lysine (K), glutamic acid (E), glutamine (Q), serine (S), or asparagine (N),
a substitution of leucine at position 65 (L65) with lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), or serine (S),
a substitution of cysteine at position 72 (C72) with serine (S),
a substitution of leucine at position 78 (L78) with serine (S), arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), glutamine (Q), or asparagine (N),
a substitution of valine at position 106 (V106) with alanine (A), aspartic acid (D), glutamic acid (E), glutamine (Q), or asparagine (N), and
a substitution of leucine at position 113 (L113) with threonine (T), arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), or serine (S).

14. The conjugate of claim 12 , wherein the p16 protein variant comprises SEQ ID NO: 2.

15. A method of intracellular delivery of a bioactive molecule in a subject, the method comprising administering the conjugate of claim 11 to the subject.

16. The method of claim 15 , wherein the bioactive molecule is a p16 protein variant comprising SEQ ID NO: 1 with an amino acid substitution at one or more of positions 15 (W15), 37 (L37), 65 (L65), 72 (C72), 78 (L78), 106 (V106), and 113 (L113) independently with lysine (K), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), serine (S), alanine (A), threonine (T), or arginine (R).

17. The method of claim 16 , wherein the amino acid substitution is at least one selected from the group consisting of:
a substitution of tryptophan at position 15 (W15) with lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), or serine (S),
a substitution of leucine at position 37 (L37) with aspartic acid (D), arginine (R), lysine (K), glutamic acid (E), glutamine (Q), serine (S), or asparagine (N),
a substitution of leucine at position 65 (L65) with lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), or serine (S),
a substitution of cysteine at position 72 (C72) with serine (S),
a substitution of leucine at position 78 (L78) with serine (S), arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), glutamine (Q), or asparagine (N),
a substitution of valine at position 106 (V106) with alanine (A), aspartic acid (D), glutamic acid (E), glutamine (Q), or asparagine (N), and
a substitution of leucine at position 113 (L113) with threonine (T), arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), or serine (S).

18. The method of claim 16 , wherein the p16 protein variant comprises SEQ ID NO: 2.

19. A method of treating breast cancer in a subject, comprising administering the conjugate of claim 12 to a subject in need of breast cancer treatment.

20. The method of claim 19 , wherein the amino acid substitution is at least one selected from the group consisting of:
a substitution of tryptophan at position 15 (W15) with lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), or serine (S),
a substitution of leucine at position 37 (L37) with aspartic acid (D), arginine (R), lysine (K), glutamic acid (E), glutamine (Q), serine (S), or asparagine (N),
a substitution of leucine at position 65 (L65) with lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), or serine (S),
a substitution of cysteine at position 72 (C72) with serine (S),
a substitution of leucine at position 78 (L78) with serine (S), arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), glutamine (Q), or asparagine (N),
a substitution of valine at position 106 (V106) with alanine (A), aspartic acid (D), glutamic acid (E), glutamine (Q), or asparagine (N), and
a substitution of leucine at position 113 (L113) with threonine (T), arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), or serine (S).

Assignees

• Samsung Electronics Co Ltd

Inventors

• Lee Jae Il
• Ahn Soshin
• Kim Jung Min
• Lee Jungmin
• Lee Jung-Hoon

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K16/30 Primary

  from tumour cells · CPC title

• A61K47/6851

  the antibody targeting a determinant of a tumour cell · CPC title

• C07K2318/20

  Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics · CPC title