Activatable interleukin 12 polypeptides
The disclosure features fusion proteins that are conditionally active variants of IL-12. In one aspect, the full-length polypeptides of the invention have reduced or minimal cytokine-receptor activating activity even though they contain a functional cytokine polypeptide. Upon activation, e.g., by cleavage of a linker that joins a blocking moiety, e.g., a steric blocking polypeptide, in sequence to the active cytokine, the cytokine can bind its receptor and effect signaling.
1. A fusion polypeptide of the formula:
[A]-[L1]-[D]-[L2]-[B] or [B]-[L1]-[A]-[L1]-[D], wherein,
A is an interleukin 12 (IL-12) polypeptide;
B is a half-life extension element, wherein the half-life extension element is human serum albumin or an antigen-binding polypeptide that binds human serum albumin;
L1 is a protease-cleavable polypeptide linker, L2 is a polypeptide linker that is optionally protease-cleavable, and when protease-cleavable L2 comprises at least one sequence that is cleavable by a protease, wherein for each of L1 and L2, independently, the protease is selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), a fibroblast activation protein (FAP), an ADAM metalloproteinase, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease; and
D is an IL-12 blocking moiety, wherein the blocking moiety is an antibody or antigen-binding fragment of an antibody that binds the IL-12 polypeptide; and
wherein
the fusion polypeptide has attenuated IL-12-receptor activating activity, wherein the IL-12-receptor activating activity of the fusion polypeptide is at least about 10 fold less than the IL-12-receptor activating activity of the polypeptide that comprises the IL-12 polypeptide that is produced by cleavage of the protease-cleavable polypeptide linker L1 or, when L2 is protease-cleavable, by cleavage of both L1 and L2, and wherein the IL-12-receptor activating activity is assessed using a HEK Blue reporter cell assay, with equal amounts on a mole basis of the IL-12 polypeptide and the fusion polypeptide.
2. The fusion polypeptide of claim 1 , wherein A comprises the formula:
[A1]-[L3]-[A2] or [A2]-[L3]-[A1], wherein
A1 is an IL-12 p40 subunit polypeptide;
A2 is an IL-12 p35 subunit polypeptide; and
L3 is a polypeptide linker that is optionally protease cleavable.
3. The fusion polypeptide of claim 1 , wherein the antibody fragment that binds the IL-12 polypeptide is a single domain antibody, Fab or scFv.
4. The fusion polypeptide of claim 1 , wherein L2 is a protease-cleavable polypeptide linker.
5. The fusion polypeptide of claim 1 , wherein L1 or L2 or both L1 and L2 are cleaved by two or more different proteases.
6. The fusion polypeptide of claim 1 , wherein the IL-12 blocking moiety inhibits activation of the IL-12 receptor by the fusion polypeptide.
7. The fusion polypeptide of claim 1 , wherein the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G.
8. The fusion polypeptide of claim 1 , wherein the matrix metalloprotease (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14.
9. A nucleic acid encoding the polypeptide of claim 1 .
10. An isolated vector comprising the nucleic acid of claim 9 .
11. The isolated host cell comprising the vector of claim 10 .
12. A method of making a pharmaceutical composition, comprising culturing the isolated host cell of claim 11 under suitable conditions for expression and collection of desired polypeptides.