Activatable antibodies that bind interleukin-6 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 interleukin-6 receptor (IL-6R), and to methods of making and using these anti-IL-6R activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.
1. An activatable antibody that in an activated state binds an Interleukin-6 Receptor (IL-6R) comprising:
an antibody or an antigen binding fragment thereof (AB) that specifically binds to the IL-6R, wherein the AB comprises (a) a heavy chain variable region comprising the variable region of the heavy chain amino acid sequence of SEQ ID NO: 1, and (b) a light chain variable region comprising a VL CDR1 sequence that comprises the VL CDR1 sequence of SEQ ID NO: 2, a VL CDR2 sequence that comprises the VL CDR2 sequence of SEQ ID NO: 2, and a VL CDR3 sequence that comprises the VL CDR3 sequence of SEQ ID NO: 2;
a masking moiety (MM) linked to the CM that inhibits the binding of the AB to the IL-6R in an uncleaved state; and
a cleavable moiety (CM) linked 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 AB comprises a VH CDR1 sequence that comprises the amino acid sequence SDHAWS (SEQ ID NO: 175); a VH CDR2 sequence that comprises the amino acid sequence YISYSGITTYNPSLKSRVT (SEQ ID NO: 176); a VH CDR3 sequence that comprises the amino acid sequence SLARTTAMDY (SEQ ID NO: 177); a VL CDR1 sequence that comprises the amino acid sequence RASQDISS (SEQ ID NO: 178); a VL CDR2 sequence that comprises the amino acid sequence TISSLQP (SEQ ID NO: 179); and a VL CDR3 sequence that comprises the amino acid sequence QQGNTLPY (SEQ ID NO: 180).
3. The activatable antibody of claim 1 , wherein the AB comprises a light chain variable region comprising the variable region of the light chain amino acid sequence SEQ ID NO: 2.
4. The activatable antibody of claim 1 , wherein the AB comprises a heavy chain variable region comprising the variable region of the heavy chain amino acid sequence SEQ ID NO: 1 and a light chain variable region comprising the variable region of the light chain amino acid sequence SEQ ID NO: 2.
5. The activatable antibody of claim 1 , wherein the AB comprises a heavy chain amino acid sequence comprising SEQ ID NO: 1 and a light chain amino acid sequence comprising SEQ ID NO: 2.
6. The activatable antibody of claim 1 , wherein the activatable antibody has the structural arrangement from N-terminus to C-terminus as follows in the uncleaved state: MM-CM-AB or AB-CM-MM.
7. The activatable antibody of claim 1 , wherein the activatable antibody comprises a linking peptide between the MM and the CM.
8. The activatable antibody of claim 1 , wherein the activatable antibody comprises a linking peptide between the CM and the AB.
9. 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 has the structural arrangement from N-terminus to C-terminus as follows in the uncleaved state: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
10. The activatable antibody of claim 9 , wherein the two linking peptides need not be identical to each other.
11. The activatable antibody of claim 9 , 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: 93) and (GGGS) n (SEQ ID NO: 94), where n is an integer of at least one.
12. The activatable antibody of claim 9 , wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 95), GGSGG (SEQ ID NO: 96), GSGSG (SEQ ID NO: 97), GSGGG (SEQ ID NO: 98), GGGSG (SEQ ID NO: 99), and GSSSG (SEQ ID NO: 100).
13. The activatable antibody of claim 9 , wherein LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 101), GSSGGSGGSGG (SEQ ID NO: 112), GSSGGSGGSGGS (SEQ ID NO: 113), GSSGGSGGSGGSGGGS (SEQ ID NO: 169), GSSGGSGGSG (SEQ ID NO: 170), or GSSGGSGGSGS (SEQ ID NO: 171).
14. The activatable antibody of claim 9 , wherein LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 172), GSSGT (SEQ ID NO: 102) or GSSG (SEQ ID NO: 103).
15. The activatable antibody of claim 9 , wherein the AB has an equilibrium dissociation constant of about 100 nM or less for binding to the IL-6R.
16. 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, and a scAb.
17. The activatable antibody of claim 1 , wherein the activatable antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-32, 109-111, 163-168, 181, and 182.
18. The activatable antibody of claim 1 , wherein the activatable antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-32, and 163-168.
19. The activatable antibody 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 the IL-6R.
20. The activatable antibody of claim 1 , wherein the MM does not interfere or compete with the AB for binding to the IL-6R in a cleaved state.
21. The activatable antibody of claim 1 , wherein the MM is a polypeptide of up to 40 amino acids in length.
22. The activatable antibody of claim 1 , wherein the MM polypeptide sequence is different from that of the IL-6R.
23. The activatable antibody of claim 1 , wherein the MM polypeptide sequence is no more than 50% identical to the IL-6R.
24. The activatable antibody of claim 1 , wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-59, 63-79, and 83-89.
25. The activatable antibody of claim 1 , wherein the protease is co-localized with the IL-6R in a tissue, and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.
26. The activatable antibody of claim 1 , wherein the CM is a polypeptide of up to 15 amino acids in length.
27. The activatable antibody of claim 1 , wherein the CM is a substrate for an enzyme selected from the group consisting of a matrix metalloprotease (MMP), thrombin, a neutrophil elastase, a cysteine protease, legumain, matriptase, and uPA.
28. The activatable antibody of claim 1 , wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 90-92, 104, 105, 107, 116-128, 157-162, 173, 174, and 183-193.
29. The activatable antibody of claim 1 , wherein the activatable antibody comprises a spacer, wherein the spacer is joined directly to the MM, and the activatable antibody has the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.
30. The activatable antibody of claim 1 comprising an agent conjugated to the AB.
31. The activatable antibody of claim 30 , wherein the agent is a therapeutic agent, an antineoplastic agent, or a toxin or fragment thereof.
32. The activatable antibody of claim 30 , wherein the agent is conjugated to the AB via a linker.
33. The activatable antibody of claim 32 , wherein the linker is a cleavable linker.
34. The activatable antibody of claim 1 , wherein the activatable antibody comprises a detectable moiety.
35. The activatable antibody of claim 34 , wherein the detectable moiety is a diagnostic agent.