Fusion protein comprising granzyme B and use thereof

1. A fusion protein comprising

(1) granzyme B; or a fragment of granzyme B comprising an active region of granzyme B and a dipeptidyl peptidase I (DPPI) cleavage sequence or an auto-cleavage sequence,

(2) a cell penetrating peptide,

(3) a targeting moiety, and

(4) a peptidase or protease cleavage site between the cell penetrating peptide and the targeting moiety,

wherein the cell penetrating peptide is a fusion peptide comprising

(1) a hydrophobic peptide comprising SEQ ID NO: 12 or a peptide fragment comprising 7 to 16 contiguous amino acid residues of SEQ ID NO: 12; and

(2) a basic peptide comprising a basic peptide unit, wherein the basic peptide unit comprises about 1 to about 6 basic amino acids or a repeat comprising about 2 to about 6 basic peptide units, wherein the basic amino acids are selected from the group consisting of lysine, arginine, and histidine;

or

(1) a hydrophobic peptide comprising a total of about 5 to about 40 amino acids, wherein the hydrophobic peptide comprises about 70% or more hydrophobic amino acids based on the total number of amino acids in the hydrophobic peptide, and wherein the hydrophobic amino acids are selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, and phenylalanine; and

(2) a basic peptide comprising KKKRK (SEQ ID NO: 15), KKKR(SEQ ID NO: 16), RKRK(SEQ ID NO: 17), RKRKRK(SEQ ID NO: 18), KKKKK(SEQ ID NO: 19), KKKKKR(SEQ ID NO: 20), KKKRKR(SEQ ID NO: 21), RRRRR(SEQ ID NO: 22), or RRRRRR(SEQ ID NO: 23).

2. The fusion protein of claim 1 , wherein the cell penetrating peptide is a fusion peptide comprising

(1) a hydrophobic peptide comprising SEQ ID NO: 12 or a peptide fragment comprising 7 to 16 contiguous amino acid residues of SEQ ID NO: 12; and

(2) a basic peptide comprising KKKRK (SEQ ID NO: 15), KKKR(SEQ ID NO: 16), RKRK(SEQ ID NO: 17), RKRKRK(SEQ ID NO: 18), KKKKK(SEQ ID NO: 19), KKKKKR(SEQ ID NO: 20), KKKRKR(SEQ ID NO: 21), RRRRR(SEQ ID NO: 22), or RRRRRR(SEQ ID NO: 23).

3. The fusion protein of claim 1 , wherein the cleavage sequence in the fragment of granzyme B comprises from about 2 to about 20 amino acids, comprising the contiguous amino acids GE or IEPD (SEQ ID NO: 36).

4. The fusion protein of claim 1 , wherein the cleavage site of the peptidase or protease is a cleavage site recognized by matrix metalloproteinase, cathepsin, urokinase-type plasminogen activator, or a combination thereof.

5. The fusion protein of claim 1 , wherein the targeting moiety comprises an antibody, an antigen-binding fragment of an antibody, a DARPin, an affibody, a lasso scaffold, a cyclotide, a knottin, an avimer, a Kunitz domain, an anticalin, an adnectin, a pronectin, a fynomer, a nanofitin, an affilin, or a combination thereof, wherein the targeting moiety targets:

a growth factor

a receptor tyrosine kinase protein, or a combination thereof.

6. The fusion protein of claim 5 , wherein the growth factor comprises epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or a combination thereof; and the receptor tyrosine kinase protein comprises an ErbB family member, insulin receptor, platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), tropomyosin-receptor-kinase (Trk) receptor, Ephrin (Eph) receptor, AXL receptor, leukocyte receptor tyrosine kinase (LTK) receptor, TIE receptor, receptor tyrosine kinase-like orphan (ROR) receptor, discoidin domain receptor (DDR), RET receptor, KLG receptor, related to receptor tyrosine kinase (RYK) receptor, Muscle-Specific Kinase (MuSK) receptor, or a combination thereof.

7. The fusion protein of claim 1 , wherein the fusion protein comprises in order from N-terminus to C-terminus (1) the granzyme B or a fragment thereof, (2) the cell penetrating peptide, (3) the cleavage site of a peptidase or protease, and (4) the targeting moiety.

8. The fusion protein of claim 1 , wherein the fusion protein comprises SEQ ID NO: 1, 3, 5, 7, or 9.

9. A pharmaceutical composition comprising the fusion protein of claim 1 and a carrier.

10. A polynucleotide encoding a fusion protein of claim 1 .

11. A method of preparing a fusion protein of claim 1 comprising expressing a polynucleotide encoding the fusion protein in a cell.