Proproteins and methods of use thereof

Abstract

The present disclosure provides for proprotein and activatable proprotein compositions. A proprotein contains a functional protein (i.e. a full length protein or functional fragment thereof) which is coupled to a peptide mask that inhibits the binding of the functional protein to its target or binding partner. An activatable proprotein contains a functional protein coupled to a peptide mask, and further coupled to an activatable linker, wherein in an non-activated state, the peptide mask inhibits binding of the functional protein to its target or binding partner and in an activated state the peptide mask does not inhibit binding of the functional protein to its target or binding partner. Proproteins can provide for reduced toxicity and adverse side effects that could otherwise result from binding of a functional protein at non-treatment sites if it were not inhibited from binding its binding partner. Proproteins can further provide improved biodistribution characteristics. Proproteins containing a peptide mask can display a longer in vivo or serum half-life than the corresponding functional protein not containing a peptide mask. The disclosure further provides methods of screening for, making, and using these proproteins.

1 - 79 . (canceled) 
     
     
         80 . An isolated activatable polypeptide comprising:
 (a) a functional protein that specifically binds to a binding partner, wherein:
 (i) the functional protein is not an antibody or an antibody fragment; 
 (ii) the functional protein is a soluble Notch receptor protein; and 
 (iii) the binding partner is a natural ligand for the soluble Notch receptor protein; 
   (b) a mask coupled to the functional protein, wherein the mask does not have an amino acid sequence of the binding partner; and   (c) a cleavable linker linked to the functional protein,   wherein:
 (i) when the activatable polypeptide is an uncleaved state, the peptide mask inhibits binding of the functional protein to its binding partner, and 
 (ii) when the activatable polypeptide is a cleaved state, the peptide mask does not inhibit binding of the functional protein to its binding partner. 
   
     
     
         81 . The isolated activatable polypeptide of  claim 80 , wherein the mask is a peptide mask. 
     
     
         82 . The isolated activatable polypeptide of  claim 80 , wherein the mask has one or more characteristics selected from:
 (a) the mask is unique for the functional protein;   (b) the mask has a therapeutic effect once uncoupled from the functional protein;   (c) the mask is 8-15 amino acids in length;   (d) the mask has less than 50% amino acid sequence homology to the natural binding partner of the functional protein;   (e) the mask contains less than 50% genetically non-encoded amino acids;   (f) the mask contains less than 50% genetically non-encoded amino acids, wherein the genetically non-encoded amino acids are D-amino acids, β-amino acids, or γ-amino acids;   (g) the mask inhibits binding of the functional protein to its binding partner allosterically;   (h) the mask inhibits binding of the functional protein to its binding partner sterically;   (i) the binding affinity of the mask to the functional protein is less than the binding affinity of the binding partner to the functional protein;   (j) the dissociation constant (K d ) of the mask towards the functional protein is at least 100 times greater than the K d  of the functional protein towards its binding partner; and   (k) the K d  of the mask towards the functional protein is lower than about 5 nM.   
     
     
         83 . The isolated activatable polypeptide of  claim 80 , wherein the mask has one or more characteristics selected from:
 (a) the mask inhibits the binding of the soluble receptor to its ligand;   (b) the mask inhibits the soluble receptor's ligand binding domain; and   (c) the mask comprises an amino acid sequence having at least 90% homology to an amino acid sequence selected from SEQ ID NOs: 56 to 77, or an amino acid sequence selected from SEQ ID NOs: 56 to 77.   
     
     
         84 . The isolated activatable polypeptide of  claim 80 , wherein the mask comprises an amino acid sequence having at least 90% homology to an amino acid sequence selected from SEQ ID NOs: 56 to 77. 
     
     
         85 . The isolated activatable polypeptide of  claim 80 , wherein the mask comprises an amino acid sequence selected from SEQ ID NOs: 56 to 77. 
     
     
         86 . The isolated activatable polypeptide of  claim 80 , wherein:
 (a) when the isolated activatable polypeptide is not in the presence of an enzyme that cleaves the cleavable linker, the mask inhibits the binding of the functional protein to the binding partner by at least 90%; and   (b) when the isolated activatable polypeptide is in the presence of the enzyme that cleaves the cleavable linker, the mask does not inhibit the binding of the functional protein to the binding partner.   
     
     
         87 . The isolated activatable polypeptide of  claim 80 , where the cleavable linker includes one or more substrates selected from:
 (a) a sequence that is susceptible to specific cleavage by an enzyme, a bond that is susceptible to reductive cleavage by a reducing agent; and   (b) a bond that is susceptible to photolytic cleavage.   
     
     
         88 . The isolated activatable polypeptide of  claim 80 , wherein the cleavable linker is a substrate for an enzyme selected from: matriptase; plasmin; MMP-9; uPA; HCV-NS3/4; PSA; and legumain. 
     
     
         89 . The isolated activatable polypeptide of  claim 80 , wherein the cleavable linker comprises an amino acid sequence VHMPLGFLGP (SEQ ID NO: 3). 
     
     
         90 . The isolated activatable polypeptide of  claim 80 , wherein the cleavable linker comprises an amino acid sequence QGPMFKSLWD (SEQ ID NO: 4). 
     
     
         91 . The isolated activatable polypeptide of  claim 80 , wherein the cleavable linker is specifically cleaved by an enzyme at a rate of at least 5×104 M−1s−1. 
     
     
         92 . The isolated activatable polypeptide of  claim 80 , further comprising at least one flexible linker. 
     
     
         93 . The isolated activatable polypeptide of  claim 92 , wherein at least one of the at least one flexible linkers is a glycine-serine rich linker. 
     
     
         94 . The isolated activatable polypeptide of  claim 93 , wherein the glycine-serine rich linker is selected from: GQSGQ (SEQ ID NO: 88); GSSGGS (SEQ ID NO: 89); GGS (SEQ ID NO: 90);
 GGGSGGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 91); and GG (SEQ ID NO: 92).   
     
     
         95 . The isolated activatable polypeptide of  claim 92 , wherein at least one of the at least one flexible linkers is positioned between the mask and the functional protein. 
     
     
         96 . The isolated activatable polypeptide of  claim 92 , wherein at least one of the at least one flexible linkers is positioned between the cleavable linker and the mask. 
     
     
         97 . The isolated activatable polypeptide of  claim 92 , wherein at least one of the at least one flexible linkers is positioned between the cleavable linker and the functional protein. 
     
     
         98 . The isolated activatable polypeptide of  claim 92 , wherein at a first of the at least one flexible linkers is positioned between the functional protein and the cleavable linker, and a second of the at least one flexible linkers is positioned between the cleavable linker and the mask. 
     
     
         99 . The isolated activatable polypeptide of  claim 92 , having an arrangement from N-terminus to C-terminus of:
 (a) [peptide mask]-flexible linker L1-[cleavable linker]-flexible linker L2-[functional protein]; or   (b) [functional protein]-flexible linker L1-[cleavable linker]-flexible linker L2-[peptide mask].   
     
     
         100 . The isolated activatable polypeptide of  claim 80 , wherein the soluble Notch receptor protein is Notch 1, Notch2, Notch3, or Notch4. 
     
     
         101 . The isolated activatable polypeptide of  claim 80 , wherein the binding partner is DLL1, DLL3, DLL4, Jagged1, or Jagged2. 
     
     
         102 . The isolated activatable polypeptide of  claim 80 , further comprising an Fc region of an immunoglobulin. 
     
     
         103 . The isolated activatable polypeptide of  claim 80 , wherein the activatable polypeptide is recombinantly expressed. 
     
     
         104 . A vector encoding the activatable polypeptide of  claim 80 . 
     
     
         105 . A pharmaceutical composition, comprising a therapeutically effective amount of the isolated activatable polypeptide of  claim 80 . 
     
     
         106 . A method of treating a disease or disorder, comprising administering a therapeutically effective amount of the pharmaceutical composition of  claim 105  to a subject in need thereof. 
     
     
         107 . The method of  claim 106 , wherein the disease or disorder is cancer. 
     
     
         108 . A method of inhibiting angiogenesis in a mammalian subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of  claim 105  to the subject.