Separation moieties and methods of use thereof

Abstract

Provided herein are separation moieties that are suitable for use in conjunction with a variety of therapeutic payloads. The separation moieties serve to generate conditionally active macromolecules whereby the macromolecules have reduced or minimal biological activity until the separation moieties are modified under specific conditions.

1 - 74 . (canceled) 
     
     
         75 . A polypeptide comprising Formula I:
   [D1]-[L1]-[D2],   wherein D1 is a first domain of interest;   L1 is a separation moiety that connects or links D1 to D2, wherein the separation moiety comprises an amino acid sequence selected from SEQ ID NOs: 195-220 or an amino acid sequence that has at least about 75% identity to SEQ ID NOs:195-220; and   D2 is a second domain of interest.   
     
     
         76 . The polypeptide of claim  1 , wherein the separation moiety comprises an amino acid sequence that is a substrate for at least one protease present in a tumor microenvironment of a human tumor. 
     
     
         77 . The polypeptide of claim  1 , comprising two or more separation moieties, wherein the two or more separation moieties are each a substrate for a protease and, wherein each separation moiety independently comprises an amino acid sequence selected from SEQ ID NOs: 195-220 or an amino acid sequence that has at least about 75% identity to SEQ ID NOs:195-220. 
     
     
         78 . The polypeptide of claim  1 , further comprising a non-cleavable linker sequence. 
     
     
         79 . The polypeptide of claim  1 , wherein [D1] or [D2] comprises a cytokine, chemokine, growth factor, a soluble receptor, or a fragment thereof, or any combination thereof. 
     
     
         80 . The polypeptide of claim  1 , wherein the polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. 
     
     
         81 . The polypeptide of claim  1 , wherein [D1] or [D2] comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor (TCR) subunit, or a fragment thereof. 
     
     
         82 . The polypeptide of claim  1 , wherein [D1] or [D2] comprises an antigen-binding polypeptide, an antibody or an antigen-binding portion thereof. 
     
     
         83 . The polypeptide of claim  1 , wherein [D1] or [D2] comprises a half-life extension domain. 
     
     
         84 . The polypeptide of claim  1 , wherein the cleavable moiety is cleaved with either (a) greater catalytic efficiency, (b) greater specificity, or (c) both (a) and (b), by one or more proteases than a reference polypeptide sequence. 
     
     
         85 . The polypeptide of claim  1 , wherein the cleavable moiety is cleaved with reduced catalytic efficiency by one or more proteases than a reference polypeptide sequence. 
     
     
         86 . The polypeptide of claim  1 , wherein the cleavable moiety is cleaved with reduced catalytic efficiency by one or more serum proteases, one or more hepatic proteases, or a protease present in a normal healthy tissue. 
     
     
         87 . A recombinant pro-protein comprising:
 a. a recombinant polypeptide comprising a cleavable moiety that is a substrate for a protease, wherein the cleavable moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220 or an amino acid sequence at least at least 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220; and   b. a polypeptide with biological activity,   
       wherein the pro-protein has attenuated biological activity, and wherein cleavage of the cleavable moiety by the protease produces a polypeptide with biological activity that is not attenuated. 
     
     
         88 . The recombinant pro-protein of  claim 87 , wherein the polypeptide with biological activity comprises a cytokine, chemokine, growth factor, a soluble receptor or a combination thereof. 
     
     
         89 . The recombinant pro-protein of  claim 87 , wherein the polypeptide with biological activity comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. 
     
     
         90 . The recombinant pro-protein of  claim 87 , wherein the polypeptide with biological activity comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor (TCR) subunit. 
     
     
         91 . The recombinant pro-protein of  claim 87 , wherein the polypeptide with biological activity comprises an antigen-binding polypeptide, an antibody or an antigen-binding portion thereof. 
     
     
         92 . A fusion protein comprising:
 a. a signaling protein or molecule;   b. a blocking moiety selected from a steric blocking moiety, a specific blocking moiety, and the combination thereof; and   c. a peptide linker that comprises a cleavable moiety that is a substrate for a protease, wherein the cleavable moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220 or an amino acid sequence at least at least 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220, wherein (a) and (b) are operably linked by (c).   
     
     
         93 . The fusion protein of  claim 92 , wherein the steric blocking moiety comprises human serum albumin (HSA), an anti-HSA antibody, an immunoglobulin Fc, or a fragment thereof.