Separation moieties and methods of use thereof
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. 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 the amino acid sequence of SEQ ID NO: 198 or a functional variant of SEQ ID NO: 198 in which one or two amino acids are substituted in SEQ ID NO: 198; and
b. a polypeptide with biological activity.
2. The recombinant pro-protein of claim 1 , wherein the polypeptide with biological activity comprises a cytokine, chemokine, growth factor, a soluble receptor, antigen-binding polypeptide, an antibody or an antigen-binding portion thereof, or a combination thereof.
3. The recombinant pro-protein of claim 1 , wherein the cleavable moiety that is a substrate for a protease comprises SEQ ID NO: 198 in which one or two amino acids are substituted in SEQ ID NO: 198 with the proviso that the amino acid at positions 4 and 5 of SEQ ID NO: 198 are not substituted.
4. The recombinant pro-protein of claim 1 , wherein the cleavable moiety that is a substrate for a protease links the polypeptide with biological activity to another amino acid sequence.
5. The recombinant pro-protein of claim 1 , 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.
6. The recombinant pro-protein of claim 1 , further comprising a blocking moiety selected from a steric blocking moiety, a specific blocking moiety, and a combination thereof.
7. The recombinant pro-protein of claim 6 , wherein the blocking moiety comprises a steric blocking moiety that comprises human serum albumin (HSA), an anti-HSA antibody, an immunoglobulin Fc, or a fragment of any of the foregoing.
8. The recombinant pro-protein of claim 6 , wherein the blocking moiety comprises a specific blocking moiety that comprises an antibody that has binding specificity for the biologically active polypeptide or an antigen-binding fragment thereof, or the ligand-binding portion of a receptor that has binding specificity for the biologically active polypeptide or a ligand-binding fragment thereof.
9. The recombinant pro-protein of claim 1 , further comprising a half-life extension domain.
10. The recombinant pro-protein of claim 1 , wherein the polypeptide with biological activity comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain.
11. The recombinant pro-protein of claim 1 , wherein the polypeptide with biological activity comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor (TCR) subunit.
12. The recombinant pro-protein of claim 1 , wherein the cleavable moiety comprises the amino acid sequence of SEQ ID NO: 198 in which one amino acid is substituted.
13. The recombinant pro-protein of claim 1 , wherein the cleavable moiety comprises the amino acid sequence of SEQ ID NO: 198 in which two amino acids are substituted.
14. The recombinant pro-protein of claim 1 , wherein the one or two amino acid substitutions in the functional variant of SEQ ID NO: 198 are conservative substitutions.
15. The recombinant pro-protein of claim 1 , wherein the functional variant of SEQ ID NO: 198 comprises the amino acid sequence of SEQ ID NO: 199, SEQ ID NO: 370, SEQ ID NO: 381, SEQ ID NO: 382, SEQ ID NO: 383, or SEQ ID NO: 390.
16. The recombinant pro-protein of claim 1 , wherein the functional variant of SEQ ID NO: 198 is a substrate for Cathepsin L.