Modified antibody containing the cleavable peptide with the amino acid sequence TGRGPSWV
The present disclosure provides modified antibodies which contain an antibody or antibody fragment (AB) modified with a masking moiety (MM). Such modified antibodies can be further coupled to a cleavable moiety (CM), resulting in activatable antibodies (AAs), wherein the CM is capable of being cleaved, reduced, photolyzed, or otherwise modified. AAs can exhibit an activatable conformation such that the AB is more accessible to a target after, for example, removal of the MM by cleavage, reduction, or photolysis of the CM in the presence of an agent capable of cleaving, reducing, or photolyzing the CM. The disclosure further provides methods of making and using such modified antibodies and activatable antibodies.
1. A cleavable polypeptide comprising amino acid sequence TGRGPSWV (SEQ ID NO: 267), wherein the amino acid sequence is cleavable by a urokinase-type plasminogen activator (uPA).
2. The cleavable polypeptide of claim 1 , wherein the polypeptide comprises an antibody or antigen binding fragment thereof (AB) that binds a target.
3. The cleavable polypeptide of claim 2 , wherein the cleavable polypeptide is linked at a position that is at or near the N-terminus of the AB.
4. The cleavable polypeptide of claim 2 , wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab′)2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
5. The cleavable polypeptide of claim 3 , wherein the AB is linked directly to the cleavable polypeptide.
6. The cleavable polypeptide of claim 3 , wherein the AB is linked to the cleavable polypeptide via a linking peptide.
7. The cleavable polypeptide of claim 2 , wherein the polypeptide comprises a masking moiety (MM).
8. The cleavable polypeptide of claim 7 , wherein the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB for binding to the target.
9. The cleavable polypeptide of claim 7 , wherein the MM is a polypeptide of no more than 40 amino acids in length.
10. The cleavable polypeptide of claim 7 , wherein the amino acid sequence of the MM is different from that of the target and is no more than 50% identical to the amino acid sequence of a natural binding partner of the AB.
11. The cleavable polypeptide of claim 7 , wherein the MM does not interfere or compete with the AB for binding to the target in a cleaved state.
12. An activatable antibody, comprising the cleavable polypeptide of claim 1 wherein said activatable antibody comprises the following structure:
a. an antibody that specifically binds to a target comprising a heavy chain variable region and a light chain variable region; and
b. a masking moiety that inhibits the binding of the antibody to its target, wherein the masking moiety is linked to the amino terminus of the light chain or heavy chain variable region via the cleavable polypeptide.
13. The activatable antibody of claim 12 , wherein the masking moiety is linked to the amino terminus of the light chain.