Activatable cytokine polypeptides and methods of use thereof

Abstract

The disclosure features fusion proteins that are conditionally active variants of a cytokine of interest. 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. Typically, the fusion proteins further comprise an in vivo half-life extension element, which may be cleaved from the cytokine in the tumor microenvironment.

1 . A pharmaceutical composition comprising:
 (i) a fusion polypeptide comprising a cytokine polypeptide [A], a blocking moiety [D], optionally a half-life extension moiety [H] and a protease-cleavable polypeptide linker; and   (ii) a second therapeutic agent;   wherein the cytokine polypeptide and the blocking moiety and the optional half-life extension element when present are operably linked by the protease-cleavable polypeptide linker and the fusion polypeptide has attenuated cytokine receptor activating activity, wherein the cytokine-receptor activating activity of the fusion polypeptide is at least about 10× less than the cytokine receptor activating activity of the polypeptide that contains the cytokine polypeptide that is produced by cleavage of the protease cleavable linker.   
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein the cytokine polypeptide is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, TGF, interferon alpha, interferon beta, interferon gamma, TNF, TGFbeta, CXCL10, CCL19, CCL20, CCL21, or a mutein, a variant, an active fragment, or a subunit of any of the foregoing. 
     
     
         3 - 4 . (canceled) 
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the second therapeutic agent is a second fusion polypeptide comprising at least one of each of: a second cytokine polypeptide [A], a blocking moiety [D], optionally a half-life extension element [H] and a protease-cleavable polypeptide linker [L];
 wherein the cytokine polypeptide and the cytokine blocking moiety and the optional half-life extension element when present are operably linked by the protease-cleavable polypeptide linker and the fusion polypeptide has attenuated cytokine receptor activating activity, wherein the cytokine-receptor activating activity of the fusion polypeptide is at least about 10× less than the cytokine receptor activating activity of the polypeptide that contains the cytokine polypeptide that is produced by cleavage of the protease cleavable linker.   
     
     
         6 . The pharmaceutical composition of  claim 5 , wherein the second cytokine polypeptide is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, TGF, interferon alpha, interferon beta, interferon gamma, TNF, TGFbeta, CXCL10, CCL19, CCL20, CCL21, a mutein thereof and active fragments thereof. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The pharmaceutical composition of  claim 5 , wherein the first fusion polypeptide comprises a IL-2 polypeptide and the second fusion polypeptide comprises a different IL-2 polypeptide, a IL-12 polypeptide, an interferon alpha polypeptide, an interferon beta polypeptide, or a mutein, or an active fragment of any of the foregoing. 
     
     
         10 . The pharmaceutical composition of elms  claim 5 , wherein the first fusion polypeptide comprises a IL-12 polypeptide and the second fusion polypeptide comprises a different IL-12 polypeptide, a IL-2 polypeptide, an interferon alpha polypeptide, an interferon beta polypeptide, or a mutein, or an active fragment of any of the foregoing. 
     
     
         11 . The pharmaceutical composition of  claim 5 , wherein the first fusion polypeptide comprises a interferon alpha polypeptide or an interferon beta polypeptide and the second fusion polypeptide comprises a different interferon alpha polypeptide, a different interferon beta polypeptide, a IL-2 polypeptide, a IL-12 polypeptide, or a mutein, or an active fragment of any of the foregoing. 
     
     
         12 . (canceled) 
     
     
         13 . The pharmaceutical composition of  claim 1 , wherein the second therapeutic agent is an agent for treating cancer. 
     
     
         14 . The pharmaceutical composition of  claim 1 , wherein the second therapeutic agent is an immunomodulator. 
     
     
         15 . (canceled) 
     
     
         16 . The pharmaceutical composition of  claim 1 , wherein each protease-cleavable polypeptide linker independently comprises a 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), an ADAM, a FAP, a cathepsin L, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease. 
     
     
         17 . The pharmaceutical composition of  claim 1 , wherein the half-life extension element comprises a serum albumin binding domain, a serum albumin, transferrin, or immunoglobulin Fc, or fragment thereof. 
     
     
         18 . The pharmaceutical composition of  claim 1 , wherein the blocking element comprises an antibody or antigen-binding fragment of an antibody that binds to the cytokine polypeptide. 
     
     
         19 . The pharmaceutical composition of  claim 1 , wherein the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 368-371, 434-440, 453-519, 523-538, 421-430, and 539-578. 
     
     
         20 . The pharmaceutical composition of  claim 1 , wherein the second therapeutic agent is a second fusion polypeptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 368-371, 434-440, 453-519, 523-538, 421-430, and 539-578, and wherein the fusion polypeptide and the second fusion polypeptide are not the same. 
     
     
         21 - 27 . (canceled) 
     
     
         28 . A fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, and 636-646 or an amino acid sequence that has at least about 80% identity to SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, and 636-646. 
     
     
         29 - 30 . (canceled) 
     
     
         31 . A fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 368-371, 434-440, 453-519, and 523-538, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs. 368-371, 434-440, 453-519, and 523-538. 
     
     
         32 . A fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 421-430, and 539-578, or an amino acid sequence that has at least 80% identity to SEQ ID NOs. 421-430, and 539-578. 
     
     
         33 . (canceled) 
     
     
         34 . A method for treating a cancer or a viral infection associated with cancer comprising administering to a subject in need thereof the pharmaceutical composition of  claim 1 . 
     
     
         35 . A nucleic acid encoding the fusion polypeptide of  claim 28 . 
     
     
         36 . A vector comprising the nucleic acid of  claim 35 . 
     
     
         37 . A host cell comprising the vector of  claim 36 . 
     
     
         38 - 68 . (canceled) 
     
     
         69 . A method for treating a cancer or a viral infection associated with cancer comprising administering to a subject in need thereof one or more fusion polypeptides of  claim 1 . 
     
     
         70 . A method for treating a cancer or a viral infection associated with cancer comprising administering to a subject in need thereof one or more fusion polypeptides of  claim 28 . 
     
     
         71 . A method for treating a cancer or a viral infection associated with cancer comprising administering to a subject in need thereof one or more fusion polypeptides of  claim 31 . 
     
     
         72 . A method for treating a cancer or a viral infection associated with cancer comprising administering to a subject in need thereof one or more fusion polypeptides of  claim 32 . 
     
     
         73 . A nucleic acid encoding the fusion polypeptide  claim 31 . 
     
     
         74 . A vector comprising the nucleic acid of  claim 73 . 
     
     
         75 . A host cell comprising the vector of  claim 74 . 
     
     
         77 . A nucleic acid encoding the fusion polypeptide of  claim 32 . 
     
     
         78 . A vector comprising the nucleic acid of  claim 77 . 
     
     
         79 . A host cell comprising the vector of  claim 78 .