Activatable antibodies that bind epidermal growth factor receptor and methods of use thereof
The invention relates generally to activatable antibodies that include a masking moiety (MM), a cleavable moiety (CM), and an antibody (AB) that specifically binds to epidermal growth factor receptor (EGFR), and to methods of making and using these anti-EGFR activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.
1. A method of treating or delaying the progression of a cancer associated with aberrant expression or activity of Epidermal Growth Factor Receptor (EGFR) in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an activatable antibody that in an activated state binds EGFR and inhibits at least one biological activity of EGFR and/or EGFR mediated signaling, wherein said activatable antibody comprises:
an antibody or an antigen binding fragment thereof (AB) that specifically binds to EGFR, wherein the AB comprises (i) a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34 and a light chain amino acid sequence comprising SEQ ID NO: 68, or (ii) a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10 and a light chain amino acid sequence comprising SEQ ID NO: 68;
a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to EGFR, wherein the MM comprises the amino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 14); and
a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease, and wherein the CM comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 13),
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.
2. The method of claim 1 , wherein the cancer is breast cancer, colorectal cancer, gastric cancer, glioblastoma, head and neck cancer, lung cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, or skin cancer.
3. The method of claim 1 , wherein the AB is conjugated to an agent.
4. The method of claim 3 , wherein the agent is a toxin or fragment thereof.
5. The method of claim 4 , wherein the agent is selected from the group consisting of a dolastatin, an auristatin or a derivative thereof, a maytansinoid or a derivative thereof, a duocarmycin or a derivative thereof, and a calicheamicin or a derivative thereof.
6. The method 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, and a scAb.
7. The method of claim 1 , wherein the activatable antibody comprises a combination of heavy and light chains selected from the group consisting of:
(a) a heavy chain comprising amino acid sequence of SEQ ID NO: 26 and a light chain comprising the amino acid sequence of SEQ ID NO: 28;
(b) a heavy chain comprising amino acid sequence of SEQ ID NO: 2 and a light chain comprising the amino acid sequence of SEQ ID NO: 4;
(c) a heavy chain comprising amino acid sequence of SEQ ID NO: 6 and a light chain comprising the amino acid sequence of SEQ ID NO: 4;
(d) a heavy chain comprising amino acid sequence of SEQ ID NO 30 and a light chain comprising the amino acid sequence of SEQ ID NO: 28;
(e) a heavy chain comprising amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 4; and
(f) a heavy chain comprising amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 28.
8. The method of claim 1 , wherein the activatable antibody comprises a heavy chain comprising amino acid sequence of SEQ ID NO: 26 and a light chain comprising the amino acid sequence of SEQ ID NO: 28.
9. The method of claim 1 , wherein the activatable antibody comprises a heavy chain comprising amino acid sequence of SEQ ID NO: 30 and a light chain comprising the amino acid sequence of SEQ ID NO: 28.
10. The method of claim 1 , wherein the activatable antibody comprises a heavy chain comprising amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 28.
11. The method of claim 1 , wherein the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB to EGFR.
12. The method of claim 1 , wherein the MM does not interfere or compete with the AB of the activatable antibody in a cleaved state for binding to EGFR.
13. The method of claim 1 , wherein the MM is a polypeptide of no more than 40 amino acids in length.
14. The method of claim 1 , wherein the MM polypeptide sequence is different from that of EGFR and wherein the MM polypeptide sequence is no more than 50% identical to any natural binding partner of the AB.
15. The method of claim 1 , wherein the CM is a polypeptide of up to 15 amino acids in length.
16. The method 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.
17. The method of claim 1 , wherein the activatable antibody comprises a linking peptide between the MM and the CM.
18. The method of claim 1 , wherein the activatable antibody comprises a linking peptide between the CM and the AB.
19. The method 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 an 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.
20. The method of claim 19 , wherein the two linking peptides need not be identical to each other.
21. The method of claim 19 , 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: 15) and (GGGS) n (SEQ ID NO: 16), where n is an integer of at least one.
22. The method of claim 19 , wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGSG (SEQ ID NO: 19), GSGGG (SEQ ID NO: 20), GGGSG (SEQ ID NO: 21), and GSSSG (SEQ ID NO: 22).
23. The method of claim 19 , wherein LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 23).
24. The method of claim 19 , wherein LP2 comprises the amino acid sequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37).
25. The method of claim 1 , wherein the activatable antibody in an uncleaved state comprises a spacer, wherein the spacer is joined directly to the MM and has the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.
26. The method of claim 25 , wherein the spacer comprises the amino acid sequence QGQSGQ (SEQ ID NO: 38).
27. The method of claim 25 , wherein the spacer and MM comprises the amino acid sequence QGQSGQCISPRGCPDGPYVMY (SEQ ID NO: 59).
28. The method of claim 3 , wherein the agent is conjugated to the AB via a linker.
29. The method of claim 28 , wherein the linker is a cleavable linker.
30. The method of claim 5 , wherein the agent is a dolastatin.
31. The method of claim 5 , wherein the agent is an auristatin or a derivative thereof.
32. The method of claim 5 , wherein the agent is auristatin E or a derivative thereof.
33. The method of claim 5 , wherein the agent is monomethyl auristatin E (MMAE).
34. The method of claim 5 , wherein the agent is a maytansinoid or a derivative thereof.
35. The method of claim 5 , wherein the agent is a maytansinoid selected from the group consisting of maytansinoid DM1 or maytansinoid DM4.
36. The method of claim 5 , wherein the agent is a duocarmycin or a derivative thereof.
37. The method of claim 5 , wherein the agent is a calicheamicin or a derivative thereof.
38. The method of claim 19 , wherein LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 23) and LP2 comprises the amino acid sequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37).
39. The method of claim 1 , wherein the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2) such that the activatable antibody in an 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, wherein LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 23) and LP2 comprises the amino acid sequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37), and wherein the AB comprises a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34, and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 68.
40. The method of claim 1 , wherein the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2) such that the activatable antibody in an 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, wherein LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 23) and LP2 comprises the amino acid sequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37), and wherein the AB comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 26, and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 68.
41. The method of claim 1 , wherein the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2) such that the activatable antibody in an 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, wherein LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 23) and LP2 comprises the amino acid sequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37), and wherein the AB comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 30, and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 68.
42. The method of claim 1 , wherein the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2) such that the activatable antibody in an 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, wherein LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 23) and LP2 comprises the amino acid sequence GSSGT (SEQ ID NO: 24), and wherein the AB comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 34, and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 68.
43. The method of claim 1 , wherein the cancer is triple-negative breast cancer, esophageal cancer, non-small cell lung cancer, osteosarcoma, squamous cell cancer, basal cell carcinoma, or melanoma.