ACTIVATABLE INTERLEUKIN-2 POLYPEPTIDES AND METHODS OF USE THEREOF
The disclosure features fusion proteins that are conditionally active variants of IL-2. 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 comprising at least one of each of:
a) a human interleukin 2 (IL-2) polypeptide [A];
b) a half-life extension element [B];
c) an IL-2 blocking moiety [D]; and
d) a protease-cleavable polypeptide linker [L]; and
wherein the IL-2 polypeptide and the IL-2 blocking moiety are operably linked by the protease-cleavable polypeptide linker and the fusion polypeptide has attenuated IL-2-receptor activating activity, wherein the IL-2-receptor activating activity of the fusion polypeptide is at least about 10 fold less than the IL-2-receptor activating activity of the polypeptide that comprises the IL-2 polypeptide that is produced by cleavage of the protease-cleavable polypeptide linker, and wherein the serum half-life of the IL-2 polypeptide that is produced by cleavage of the protease-cleavable polypeptide linker is comparable to the half-life of naturally occurring IL-2.
2 . The fusion polypeptide of claim 1 , further comprising a tumor-specific antigen binding peptide.
3 . The fusion polypeptide of claim 2 , wherein the tumor-specific antigen binding peptide is linked to any one of [A], [B], or [D] by a non-cleavable linker.
4 . The fusion polypeptide of claim 2 , wherein the tumor-specific antigen binding peptide is linked to any one of [A], [B], or [D] by a cleavable linker.
5 . The fusion polypeptide of claim 2 , wherein the tumor-specific antigen binding peptide is linked to the IL-2 polypeptide by a non-cleavable linker and wherein the IL-2 polypeptide is linked to the half-life extension element or the IL-2 blocking moiety by a cleavable linker.
6 . The fusion polypeptide of claim 1 , wherein the IL-2-receptor activating activity is assessed using a CTLL-2 proliferation assay, a phospho STAT ELISA, or HEK Blue reporter cell assay and using equal amounts on a mole basis of the IL-2 polypeptide and the fusion polypeptide.
7 . The fusion polypeptide of claim 1 , comprising a plurality of protease-cleavable polypeptide linkers, wherein each protease-cleavable polypeptide linker independently comprises at least one sequence that is capable of being cleaved by a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, 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.
8 . The fusion polypeptide of claim 1 , wherein IL-2 blocking moiety inhibits activation of the IL-2 receptor by the fusion polypeptide.
9 . The fusion polypeptide of claim 1 , wherein the IL-2 blocking moiety comprises a ligand-binding domain or fragment of a cognate receptor for the IL-2, a single domain antibody, Fab or scFv that binds the IL-2 polypeptide, or an antibody or antibody fragment that binds a receptor of the IL-2.
10 . The fusion polypeptide of claim 1 , wherein each protease-cleavable polypeptide linker independently comprises two or more cleavage sites for the same protease, or two or more cleavage sites that are cleaved by different proteases or at least one of the protease-cleavable polypeptide linkers comprises a cleavage site for two or more different proteases.
11 . The fusion polypeptide of claim 1 , wherein the half-life extension element is human serum albumin, an antigen-binding polypeptide, which binds human serum albumin, or an immunoglobulin Fc.
12 . A nucleic acid encoding the polypeptide of claim 1 .
13 . A vector comprising the nucleic acid of claim 12 .
14 . A host cell comprising the vector of claim 13 .
15 . A method of making a pharmaceutical composition, comprising culturing the host cell of claim 14 under suitable conditions for expression and collection of the fusion polypeptide.
16 . A fusion polypeptide of the formula:
[B]-[L1]-[A]-[L1]-[D]
or
[D]-[L1]-[A]-[L1]-[B],
[A]-[L1]-[B]-[L2]-[D]
or
[A]-[L1]-[D]-[L2]-[B]
or
[D]-[L2]-[B]-[L1]-[A]
or
[B]-[L2]-[D]-[L1]-[A]
or
[D]-[L1]-[B]-[L1]-[A]
or
[B]-[L1]-[D]-[L1]-[A]
or
wherein,
A is an interleukin 2 (IL-2) polypeptide;
B is a half-life extension element;
L1 and L2 are each independently a polypeptide linker, wherein L1 is a protease-cleavable polypeptide linker and L2 is optionally a protease-cleavable polypeptide linker;
D is an IL-2 blocking moiety; and
wherein the fusion polypeptide has attenuated IL-2-receptor activating activity, wherein the IL-2-receptor activating activity of the fusion polypeptide is at least about 10 fold less than the IL-2-receptor activating activity of the polypeptide that comprises the IL-2 polypeptide that is produced by cleavage of the protease-cleavable polypeptide linker L1.
17 . The fusion polypeptide of claim 16 , wherein the IL-2 blocking moiety comprises a ligand-binding domain or fragment of a cognate receptor for the IL-2, a single domain antibody, Fab or scFv that binds the IL-2 polypeptide, or an antibody or antibody fragment that binds a receptor of the IL-2.
18 . The fusion polypeptide of claim 16 , wherein the half-life extension element is human serum albumin, an antigen-binding polypeptide, which binds human serum albumin, or an immunoglobulin Fc.
19 . A method for treating a tumor, comprising administering to a subject in need thereof an effective amount of a fusion polypeptide comprising at least one of each of:
a) an interleukin 2 (IL-2) polypeptide [A];
b) a half-life extension element [B];
c) an IL-2 blocking moiety [D]; and
d) a protease-cleavable polypeptide linker [L]; and
wherein the IL-2 polypeptide and the IL-2 blocking moiety are operably linked by the protease-cleavable polypeptide linker and the fusion polypeptide has attenuated IL-2-receptor activating activity, wherein the IL-2-receptor activating activity of the fusion polypeptide is at least about 10 fold less than the IL-2-receptor activating activity of the polypeptide that comprises the IL-2 polypeptide that is produced by cleavage of the protease-cleavable polypeptide linker.
20 . The method of claim 19 , wherein the method comprises administering effective amount of the fusion polypeptide intravenously.