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 functional protein that specifically binds to a binding partner, wherein the functional protein is not an antibody or an antibody fragment;   a peptide mask coupled to the functional protein, wherein the peptide mask does not have an amino acid sequence of the binding partner; and   a cleavable linker linked to the functional protein, wherein the cleavable linker comprises a sequence for a substrate of matriptase,   wherein when the activatable polypeptide is an uncleaved state, the peptide mask inhibits binding of the functional protein to its binding partner, and   wherein 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 activatable polypeptide of  claim 80 , wherein the functional protein is an interferon-alpha (IFN-α) functional protein, and wherein the binding partner is a receptor for the IFN-α protein. 
     
     
         82 . The activatable polypeptide of  claim 81 , wherein IFN-α functional protein is selected from the group consisting of 2a, 2b and con1. 
     
     
         83 . The activatable polypeptide of  claim 80 , wherein the sequence for the matriptase substrate is selected from the group consisting of XXQAR(A/V)X (SEQ ID NO: 87) and AGPR (SEQ ID NO: 2). 
     
     
         84 . The activatable polypeptide of  claim 80 , wherein the peptide mask has one or more of the following characteristics:
 (i) the peptide mask is unique for the functional protein;   (ii) the peptide mask has a therapeutic effect once uncoupled from the functional protein;   (iii) the peptide mask is 8-15 amino acids in length;   (iv) the peptide mask has less than 50% amino acid sequence homology to the natural binding partner of the functional protein;   (v) the peptide mask contains less than 50% genetically non-encoded amino acids;   (vi) the peptide 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;   (vii) the peptide mask inhibits binding of the functional protein to its binding partner allosterically;   (viii) the peptide mask inhibits binding of the functional protein to its binding partner sterically;   (ix) the binding affinity of the peptide mask to the functional protein is less than the binding affinity of the binding partner to the functional protein;   (x) the dissociation constant (Kd) of the peptide mask towards the functional protein is at least 10 times greater than the Kd of the functional protein towards its binding partner;   (xi) the dissociation constant (Kd) of the peptide mask towards the functional protein is at least 100 times greater than the Kd of the functional protein towards its binding partner; or   (xii) the Kd of the peptide mask towards the functional protein is lower than about 5 nM.   
     
     
         85 . The activatable polypeptide of  claim 81  wherein the peptide mask contains a sequence selected from those presented in Table 3 or a sequence having at least 90% homology thereof. 
     
     
         86 . The activatable polypeptide of  claim 80 , wherein when the activatable polypeptide is not in the presence of an enzyme that cleaves the cleavable linker, the peptide mask of the activatable polypeptide inhibits the binding of the functional protein to its binding partner by at least 90% when compared to when the composition is in the presence of the enzyme that cleaves the cleavable linker and the peptide mask does not inhibit the binding of the functional protein to its binding partner. 
     
     
         87 . The activatable polypeptide of  claim 80 , wherein when the activatable polypeptide is exposed to the matriptase enzyme, the enzyme cleaves the cleavable linker in the activatable polypeptide. 
     
     
         88 . The activatable polypeptide of  claim 80 , wherein the activatable polypeptide in the uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: (peptide mask)-(cleavable linker)-(functional protein or functional fragment thereof) or (functional protein or functional fragment thereof)-(cleavable linker)-(peptide mask). 
     
     
         89 . The activatable polypeptide of  claim 81 , wherein the IFN-α functional protein has an equilibrium dissociation constant of no more than 100 nM for binding to the receptor for the IFN-α protein. 
     
     
         90 . A pharmaceutical composition comprising a therapeutically effective amount of the activatable polypeptide according to  claim 80  and a pharmaceutically acceptable excipient. 
     
     
         91 . A method of treating a condition in a mammalian subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an activatable polypeptide or a pharmaceutical composition thereof, wherein the pharmaceutical composition comprises a therapeutically effective amount of the activatable polypeptide and a pharmaceutically acceptable excipient,
 wherein the activatable polypeptide comprises:   a functional protein that specifically binds to a binding partner is not an antibody or an antibody fragment;   a peptide mask coupled to the functional protein, wherein the peptide mask does not have an amino acid sequence of the binding partner; and   a cleavable linker linked to the functional protein, wherein the cleavable linker comprises a sequence for a substrate of matriptase,   wherein when the activatable polypeptide is an uncleaved state, the peptide mask inhibits binding of the functional protein to its binding partner, and   wherein when the activatable polypeptide is a cleaved state, the peptide mask does not inhibit binding of the functional protein to its binding partner.   
     
     
         92 . The method of  claim 91 , wherein the condition is cancer. 
     
     
         93 . The method of  claim 91 , wherein the functional protein is an interferon-alpha (IFN-α) functional protein, and wherein the binding partner is a receptor for the IFN-α protein. 
     
     
         94 . The method of  claim 93 , wherein IFN-α functional protein is selected from the group consisting of 2a, 2b and con1. 
     
     
         95 . The method of  claim 91 , wherein the sequence for the matriptase substrate is selected from the group consisting of XXQAR(A/V)X (SEQ ID NO: 87) and AGPR (SEQ ID NO: 2). 
     
     
         96 . The method of  claim 91 , wherein the peptide mask has one or more of the following characteristics:
 (i) the peptide mask is unique for the functional protein;   (ii) the peptide mask has a therapeutic effect once uncoupled from the functional protein;   (iii) the peptide mask is 8-15 amino acids in length;   (iv) the peptide mask has less than 50% amino acid sequence homology to the natural binding partner of the functional protein;   (v) the peptide mask contains less than 50% genetically non-encoded amino acids;   (vi) the peptide 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;   (vii) the peptide mask inhibits binding of the functional protein to its binding partner allosterically;   (viii) the peptide mask inhibits binding of the functional protein to its binding partner sterically;   (ix) the binding affinity of the peptide mask to the functional protein is less than the binding affinity of the binding partner to the functional protein;   (x) the dissociation constant (Kd) of the peptide mask towards the functional protein is at least 10 times greater than the Kd of the functional protein towards its binding partner;   (xi) the dissociation constant (Kd) of the peptide mask towards the functional protein is at least 100 times greater than the Kd of the functional protein towards its binding partner; or   (xii) the Kd of the peptide mask towards the functional protein is lower than about 5 nM.   
     
     
         97 . The method of  claim 93  wherein when the functional protein is an interferon-alpha (IFN-α) functional protein, and when the binding partner is a receptor for the IFN-α protein, the peptide mask contains a sequence selected from those presented in Table 3 or a sequence having at least 90% homology thereof. 
     
     
         98 . A method of manufacturing the activatable polypeptide of  claim 80 , the method comprising the steps of:
 (a) culturing a cell comprising a nucleic acid construct that encodes the activatable polypeptide under conditions that lead to expression of the activatable polypeptide, wherein the activatable polypeptide in the uncleaved state comprises; and   (b) recovering the activatable polypeptide.