Anti-EGFR activatable antibodies
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. An activatable antibody that in an activated state binds Epidermal Growth Factor Receptor (EGFR), the activatable antibody comprising:
an antibody or an antigen binding fragment thereof (AB) that specifically binds to EGFR;
a masking moiety (MM) coupled to the AB that inhibits the binding of the AB of the activatable antibody in an uncleaved state to EGFR, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 218-220, 236-265 and 266; and
at least one cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.
2. The activatable antibody of claim 1 , wherein the MM comprises the amino acid sequence CISPRGC (SEQ ID NO: 1).
3. The activatable antibody of claim 1 , wherein the MM comprises the amino acid sequence CISPRGCPDGPYVM (SEQ ID NO: 218) or CISPRGCPDGPYVMY (SEQ ID NO: 238).
4. The activatable antibody of claim 1 , wherein the MM does not interfere or compete with the AB for binding to EGFR when the activatable antibody is in a cleaved state.
5. The activatable antibody of claim 1 , wherein the MM is a polypeptide of no more than 40 amino acids in length.
6. The activatable antibody of claim 1 , wherein in the presence of EGFR, the MM reduces the ability of the AB to bind EGFR by at least 90% when the CM is uncleaved, as compared to when the CM is cleaved when assayed in vitro using a target displacement assay.
7. The activatable antibody of claim 1 , wherein the protease is co-localized with EGFR in a tissue, and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.
8. The activatable antibody of claim 1 , wherein the CM is a polypeptide of up to 15 amino acids in length.
9. The activatable antibody of claim 1 , wherein the CM is a substrate for an enzyme selected from the group consisting of the enzymes in Table 3.
10. The activatable antibody of claim 1 , wherein the CM is a substrate for an enzyme selected from the group consisting of a urokinase-type plasminogen activator (uPA), a legumain, a matriptase, and an MMP.
11. The activatable antibody of claim 1 , wherein the CM comprises an amino acid sequence selected from the group consisting of TGRGPSWV (SEQ ID NO: 267); SARGPSRW (SEQ ID NO: 268); TARGPSFK (SEQ ID NO: 269); TARGPSW (SEQ ID NO: 270), LSGRSDNH (SEQ ID NO: 271); GGWHTGRN (SEQ ID NO: 272); HTGRSGAL (SEQ ID NO: 273); PLTGRSGG (SEQ ID NO: 274); LTGRSGA (SEQ ID NO: 275); AARGPAIH (SEQ ID NO: 276); RGPAFNPM (SEQ ID NO: 277); SSRGPAYL (SEQ ID NO: 278); RGPATPIM (SEQ ID NO: 279); RGPA (SEQ ID NO: 280), GLSGRSDNHGSS (SEQ ID NO: 366); PLTGRSGGGGSS (SEQ ID NO: 367); ETPSVKTMGRSS (SEQ ID NO: 368); and GTGRGPSWVGSS (SEQ ID NO: 369).
12. The activatable antibody of claim 1 , wherein the CM comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 271).
13. The activatable antibody of claim 1 , wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM.
14. The activatable antibody of claim 1 , wherein the activatable antibody comprises a linking peptide between the MM and the CM.
15. The activatable antibody of claim 1 , wherein the activatable antibody comprises a linking peptide between the CM and the AB.
16. The activatable antibody of claim 1 , wherein the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
17. The activatable antibody of claim 16 , wherein the two linking peptides need not be identical to each other.
18. The activatable antibody of claim 16 , wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.
19. The activatable antibody of claim 16 , wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS) n , (GGS)n, (GSGGS)n (SEQ ID NO: 12) and (GGGS)n (SEQ ID NO: 13), where n is an integer of at least one.
20. The activatable antibody of claim 16 , wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 14), GGSGG (SEQ ID NO: 15), GSGSG (SEQ ID NO: 16), GSGGG (SEQ ID NO: 17), GGGSG (SEQ ID NO: 18), and GSSSG (SEQ ID NO: 19).
21. The activatable antibody of claim 16 , wherein the activatable antibody is selected from the group consisting of:
an activatable antibody wherein LP1 comprises the amino acid sequence GGSGGS (SEQ ID NO: 111);
an activatable antibody wherein LP2 comprises the amino acid sequence GS or GSSG;
and an activatable antibody wherein LP1 comprises the amino acid sequence GGSGGS (SEQ ID NO: 111) and LP2 comprises the amino acid sequence GS or GSSG.
22. The activatable antibody of claim 16 , wherein the CM is a substrate for an enzyme selected from the group consisting of a urokinase-type plasminogen activator (uPA), a legumain and a matriptase, wherein the MM comprises the amino acid sequence CISPRGC (SEQ ID NO: 1), wherein LP1 comprises the amino acid sequence GGSGGS (SEQ ID NO: 111), and wherein LP2 comprises the amino acid sequence GS or GSSG.
23. The activatable antibody of claim 1 , wherein the AB has a dissociation constant of at most 100 nM for binding to EGFR.
24. The activatable antibody of claim 1 , 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.
25. The activatable antibody of claim 1 , wherein the AB is cetuximab, panitumumab, zalutumumab, matuzumab, nimotuzumab, ICR62, mAb 528, CH806, or MDX447 or an antigen binding fragment of cetuximab, panitumumab, zalutumumab, matuzumab, nimotuzumab, ICR62, mAb 528, CH806, or MDX447.
26. The activatable antibody of claim 1 , wherein the AB is cetuximab or an antigen binding fragment of cetuximab.
27. The activatable antibody of claim 1 , wherein the activatable antibody comprises a second AB wherein the target for the second AB is selected from the group consisting of the targets in Table 1.
28. The activatable antibody of claim 1 , wherein the AB is conjugated to an agent.
29. The activatable antibody of claim 28 , wherein the agent is a therapeutic agent.
30. The activatable antibody of claim 29 , wherein the agent is an antineoplastic agent.
31. The activatable antibody of claim 28 , wherein the agent is a toxin or a fragment thereof.
32. The activatable antibody of claim 28 , wherein the agent is an agent selected from the group consisting of the agents in Table 4.
33. The activatable antibody of claim 28 , wherein the agent is conjugated to the AB via a linker.
34. The activatable antibody of claim 33 , wherein the linker is a cleavable linker.
35. The activatable antibody of claim 33 , wherein the linker is a non-cleavable linker.
36. The activatable antibody of claim 1 , wherein the activatable antibody comprises a detectable moiety.
37. The activatable antibody of claim 36 , wherein the detectable moiety is a diagnostic agent.
38. The activatable antibody of claim 1 , wherein the serum half-life of the activatable antibody is at least 5 days when administered to an organism.
39. An isolated nucleic acid molecule encoding the activatable antibody of claim 1 .
40. A vector comprising the isolated nucleic acid molecule of claim 39 .
41. A method of producing an activatable antibody by culturing a cell under conditions that lead to expression of the activatable antibody, wherein the cell comprises the vector of claim 40 .