Hepatocyte Growth Factor Fragments that Function as Potent Met Receptor Agonists and Antagonists

Abstract

The NK1 fragment of hepatocyte growth factor (HGF) binds to and activates the Met receptor, a transmembrane receptor tyrosine kinase that plays a critical role in embryonic development and organ formation. The instant application discloses NK1 variant polypeptides which act as agonists or antagonists of HGF. Further disclosed are covalently linked NK1 variant polypeptides. Many of the disclosed variant polypeptides possess improved stability characteristics.

1 . A variant of human Hepatocyte Growth Factor (hHGF) comprising at least one member selected from an amino acid substitution, an amino acid deletion, an amino acid addition and combinations thereof in the NK1 region, whereby the resulting HGF variant exhibits a characteristic which is a member selected from an affinity for Met greater than that of wild type hHGF, thermal stability greater than that of wild type hHGF, salt stability greater that that of wild-type hHGF, and a combination thereof. 
     
     
         2 . The variant according to  claim 1 , wherein said variant is an antagonist of Met. 
     
     
         3 . The variant according to  claim 1 , wherein said variant is an agonist of Met. 
     
     
         4 . The variant according to  claim 1 , wherein said variant includes an amino acid substitution which is a member selected from K137R, K170E, N193D, K62E, Q173R, F190Y, V64A, Q95R, K132N/R, N127D/A/K/R, N77S, S154A, 1130V, 1142T/V, K148E and combinations thereof. 
     
     
         5 . The variant according to  claim 1 , wherein said variant is conjugated to a member selected from a detectable moiety, a water-soluble polymer, a water-insoluble polymer, a therapeutic moiety, a targeting moiety and a combination thereof. 
     
     
         6 . The variant according to  claim 5 , wherein said variant is conjugated to a detectable moiety selected from a radioisotope, a paramagnet, a fluorophore and combinations thereof. 
     
     
         7 . The variant according to  claim 6 , wherein said variant is a diagnostic imaging agent. 
     
     
         8 . A pharmaceutical formulation comprising a variant according to  claim 1 , wherein said variant is in combination with a pharmaceutically acceptable carrier. 
     
     
         9 . A method of treating cancer in a subject in need of said treatment, said method comprising administering to said subject a therapeutically effective amount of a variant according to  claim 1 , thereby treating said cancer. 
     
     
         10 . The method according to  claim 9 , wherein said cancer is associated with Met activation. 
     
     
         11 . A method of reducing a process which is a member selected from tumor progression, angiogenesis, metastasis and combinations thereof in a subject, said method comprising administering to said subject an amount of a variant according to  claim 1  sufficient to reduce said process. 
     
     
         12 . The method according to  claim 8 , wherein said cancer is a member selected from colorectal, oral, hepatocellular, renal, breast, lung, ovarian, stomach, brain, prostate, and combinations thereof. 
     
     
         13 . A method of diagnosing a disease in a subject, said method comprising, administering to said subject an amount of a variant according to  claim 7 , and detecting said variant. 
     
     
         14 . A dimer comprising a first human NK1 (hNK1) variant polypeptide and a second hNK1 variant polypeptide, wherein the first hNK1 variant polypeptide comprises a first linker and the second hNK1 polypeptide comprises a second linker, wherein the first linker comprises at least one cysteine residue and the second linker comprises a moiety reactive with a sulfhydryl group of the cysteine, wherein the dimer is formed by the reaction of cysteine with the moiety reactive with the sulfhydryl group. 
     
     
         15 . The dimer according  claim 14 , wherein the first and the second variant hNK1 variant polypeptide contain amino acid substitutions. 
     
     
         16 . A nucleic acid encoding the variant polypeptide of  claim 14 . 
     
     
         17 . A cell wherein the nucleic acid of  claim 16  is expressed. 
     
     
         18 . A method of tissue regeneration, said method comprising contacting cells with an effective amount of the dimer of  claim 14 . 
     
     
         19 . A pharmaceutical formulation comprising the variant polypeptide of  claim 14 , wherein the variant polypeptide is in combination with a pharmaceutically acceptable carrier.