Molecular design of recombinant protein drug

Abstract

Provided is a mutant of an endostatin. The mutant has improved ATPase activity and improved activity of inhibiting angiogenesis and inhibiting tumors. Further provided is use of the mutant in treatment of angiogenesis related diseases such as tumors.

1 . A method for increasing the anti-angiogenesis activity of endostatin or variants thereof, including increasing the ATPase activity of endostatin or variants thereof. 
     
     
         2 . The method of  claim 1 , including genetic engineering on the A motif and/or the B motif and/or the C motif of endostatin or variants thereof, to obtain an endostatin mutant with increased ATPase activity. 
     
     
         3 . The method of  claim 2 , wherein said genetic engineering includes one or more of the following approaches: (1) keeping the amino acid residues corresponding to the conserved amino acid residues G89, G92, and K95 in the A motif GXXGXXK in SEQ ID NO:1 unchanged; (2) increasing the spatial conformation flexibility of the peptide corresponding to the A motif by adjusting the variable residues X within the A motif GXXGXXK; (3) adding a Ser or Thr after residue K95 in the classic sequence of A motif GXXGXXK; (4) adjusting the B motif according to the change in the A motif; (5) partially or entirely mutating the amino acid residues in the C motif; (6) adjusting the C motif according to the change in the B motif; (7) adjusting the C motif according to the change in the A motif; (8) changing the A, B, and C motifs at the same time. 
     
     
         4 . The method of  claim 2 , wherein the B motif is kept unchanged. 
     
     
         5 . The method of  claim 1 , wherein said endostatin mutant has a sequence selected from the group consisting of SEQ ID NO: 3-34 and SEQ ID NO: 37-39. 
     
     
         6 . The method of  claim 1 , wherein said endostatin mutant has a sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30. 
     
     
         7 . A mutant of endostatin or variants thereof, wherein said mutant has increased anti-angiogenesis activity, and wherein said mutant comprises a mutation in the ATP-binding motif thereof, and has increased ATPase activity compared to the corresponding wild-type endostatin or variants thereof. 
     
     
         8 . The mutant of  claim 7 , wherein said mutant contains a mutation in the sequence corresponding to the Gly-Ser-Glu-Gly-Pro-Leu-Lys motif consisting of amino acid residues at positions 89-95 of SEQ ID NO: 1, and wherein said mutation is selected from substitution, deletion, and/or addition of one or several amino acid residues, or a combination thereof, and wherein said mutation leads to increased ATPase activity of the mutant. 
     
     
         9 . The mutant of  claim 7 , wherein the sequence of said mutant corresponding to the Gly-Ser-Glu-Gly-Pro-Leu-Lys motif consisting of amino acid residues at positions 89-95 of SEQ ID NO: 1 is partially or completely deleted. 
     
     
         10 . The mutant of  claim 7 , wherein one or more amino acid residues of said mutant which correspond to amino acid residues at positions 89, 92 and 95 in SEQ ID NO:1 are partially or completely substituted or deleted. 
     
     
         11 . The mutant of  claim 7 , wherein
 (a) the amino acid residue corresponding to Gly at position 89 in SEQ ID NO:1 is deleted or substituted by an uncharged or aromatic amino acid; or   (b) the amino acid residue corresponding to Gly at position 92 in SEQ ID NO:1 is deleted or substituted by an uncharged amino acid; or   (c) the amino acid residue corresponding to Lys at position 95 in SEQ ID NO:1 is deleted or substituted by a positively charged or an uncharged amino acid; or   any combination of (a)-(c).   
     
     
         12 . The mutant of  claim 11 , wherein
 (a) the amino acid residue corresponding to Gly at position 89 in SEQ ID NO:1 is deleted or substituted by Ala or Pro; or   (b) the amino acid residue corresponding to Gly at position 92 in SEQ ID NO:1 is deleted or substituted by Ala; or   (c) the amino acid residue corresponding to Lys at position 95 in SEQ ID NO:1 is deleted or substituted by Arg or Gln; or   any combination of (a)-(c).   
     
     
         13 . The mutant of  claim 7 , wherein said mutant has a sequence selected from the group consisting of SEQ ID NO: 3-34 and SEQ ID NO: 37-39. 
     
     
         14 . The mutant of  claim 7 , wherein said mutant has a sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30. 
     
     
         15 . A pharmaceutical composition which comprises a mutant of  claim 7  and a pharmaceutically acceptable carrier. 
     
     
         16 . The pharmaceutical composition of  claim 15 , wherein said mutant is covalently linked to a PEG molecule. 
     
     
         17 . The pharmaceutical composition of  claim 16 , wherein said PEG molecule is covalently linked to the α-amino group at the N-terminal of said mutant. 
     
     
         18 . The pharmaceutical composition of  claim 16 , wherein said monomethoxypolyethylene glycol is monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD). 
     
     
         19 . A method of treating an angiogenesis-related disease, including administering to a patient an effective amount of a mutant of  claim 7 . 
     
     
         20 . The method of  claim 19 , wherein said angiogenesis-related disease is tumor, obesity, fatty liver, or insulin resistance.