Chimeric activators: quantitatively designed protein therapeutics and uses thereof

Abstract

Aspects of the invention provide methods for harnessing the potential of proteins that occur naturally (e.g., in humans) and that have serious but finite toxicity. Aspects of the invention relate to a quantitative systems-biological and structural approach to design a class of chimeric proteins that avoid the toxicity of protein drugs while retaining their desired activities. In particular, chimeric proteins containing a variant form of a natural protein fused to a targeting moiety may be administered to a subject to target a signal (e.g., induction of apoptosis) to particular cells without having a generalized toxic effect in the subject.

1 . An isolated nucleic acid encoding a chimeric activator, the nucleic acid comprising in frame a first sequence, a second sequence, and optionally a third sequence, wherein the first sequence encodes an activity element, the second sequence encodes a targeting element, and the third sequence encodes a linker, and wherein the first sequence includes a mutation that reduces the binding affinity of the activity element for a natural cell surface receptor. 
     
     
         2 . An isolated chimeric activator protein encoded by  claim 1 . 
     
     
         3 . An isolated nucleic acid of  claim 1 , wherein the first, second, and third sequences are arranged to encode a chimeric activator protein that comprises the targeting element at its N-terminal end, the activity element at its C-terminal end, and the linker connecting the C-terminal end of the targeting element to the N-terminal end of the activity element. 
     
     
         4 . An isolated nucleic acid of  claim 1 , wherein the first, second, and third sequences are arranged to encode a chimeric activator protein that comprises the activity element at its N-terminal end, the targeting element at its C-terminal end, and the linker connecting the C-terminal end of the activity element to the N-terminal end of the targeting element. 
     
     
         5 . A chimeric activator protein of  claim 2 , wherein said chimeric activator protein selectively binds to cancer cells relative to normal cells. 
     
     
         6 . A nucleic acid of  claim 1 , wherein the activity element is a cytokine. 
     
     
         7 . A nucleic acid of  claim 1 , wherein the targeting element is an antibody. 
     
     
         8 . A nucleic acid of  claim 1 , wherein the targeting element is an antibody fragment. 
     
     
         9 . A nucleic acid of  claim 1 , wherein the targeting element is an Fc region. 
     
     
         10 . A nucleic acid of  claim 1 , wherein the activity element activates apoptosis. 
     
     
         11 . A nucleic acid of  claim 1 , wherein the activity element is an interferon. 
     
     
         12 . A nucleic acid of  claim 11 , wherein the activity element is a mutant interferon 2 alpha. 
     
     
         13 . A nucleic acid of  claim 12 , wherein the activity element is interferon 2 alpha (K133A). 
     
     
         14 . A nucleic acid of  claim 12 , wherein the activity element is interferon 2 alpha (R144A). 
     
     
         15 . A nucleic acid of  claim 12 , wherein the activity element is interferon 2 alpha (R149A). 
     
     
         16 . A nucleic acid of  claim 1 , wherein the targeting element is a ligand. 
     
     
         17 . A nucleic acid of  claim 16 , wherein the targeting element is epidermal growth factor. 
     
     
         18 . An isolated nucleic acid comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, and SEQ ID NO. 15. 
     
     
         19 . An isolated protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, and SEQ ID NO. 16. 
     
     
         20 . A nucleic acid of  claim 1 , wherein the targeting element is MR1-1, a CEA-binding antibody, or a glycophorin-binding antibody. 
     
     
         21 . A nucleic acid of  claim 1  or  19 , wherein the activity element is a TRAIL or EPO activity element. 
     
     
         22 . An isolated chimeric activator protein encoded by the nucleic acid of  claim 19 . 
     
     
         23 . An isolated chimeric activator protein encoded by the nucleic acid of  claim 20 . 
     
     
         24 . A recombinant protein comprising
 a first element that binds to a first receptor on a target cell;   a second element that binds to a second receptor on the target cell; and   optionally a linker that connects the first and second elements,   wherein the linker allows the first and second elements to bind simultaneously to the first and second receptors on the target cell, wherein the first element comprises a mutation that reduces its binding affinity for its receptors, and wherein the first element transmits a signal to the host cell when it is bound to the first receptor.   
     
     
         25 . The recombinant protein of  claim 24 , wherein the first element binds to its receptor as a monomer. 
     
     
         26 . The recombinant protein of  claim 24 , wherein the second element binds to its receptor as a monomer. 
     
     
         27 . The recombinant protein of  claim 24 , wherein the linker is between about 25 and about 70 amino acids long. 
     
     
         28 . The recombinant protein of  claim 24 , wherein the linker is flexible. 
     
     
         29 . A chimeric activator comprising an activity element and a targeting element optionally joined by a linker, wherein the targeting element binds to a first binding site on a target cell, and wherein the activity element binds to a second binding site on a target cell, and wherein the linker length has been optimized for maximizing binding efficiency of the activity element to the second binding site when the targeting element is bound to the first binding site. 
     
     
         30 . The chimeric activator of  claim 29  wherein the first binding site is a ligand binding site, a receptor, or an epitope on a cell surface. 
     
     
         31 . The chimeric activator of  claim 29  wherein the second binding site is a signal-transduction receptor on a cell surface. 
     
     
         32 . The chimeric activator of  claim 29  wherein the targeting element is an antibody, an antibody fragment, an Fc region, a natural or synthetic ligand, or a protein interaction motif. 
     
     
         33 . The chimeric activator of  claim 29  wherein the activity element is a cytokine, a cytotoxic protein, a regulatory protein, or an active fragment of any one thereof. 
     
     
         34 . The chimeric activator of  claim 29  wherein the linker length is at least equal to the minimum distance between the first and second binding sites on the cell surface. 
     
     
         35 . The chimeric activator of  claim 29  wherein the linker length is at least twice the minimum distance between the first and second binding sites on the cell surface. 
     
     
         36 . The chimeric activator of  claim 29  wherein the linker length is at most five-six times the minimum distance between the first and second binding sites on the cell surface. 
     
     
         37 . The chimeric activator of  claim 29  wherein the activity element is mutated such that the binding affinity of the activity element for the second binding site is lower than the binding affinity of the targeting element for the first binding site. 
     
     
         38 . The chimeric activator of  claim 29 , wherein the activity element is an enzyme. 
     
     
         39 . A method for manufacturing a chimeric activator comprising an activity element and a targeting element joined by a linker, wherein the targeting element binds to a first binding site on a target cell, and wherein the activity element binds to a second binding site on a target cell, and wherein the linker length has been optimized for maximizing binding efficiency of the activity element to the second binding site when the targeting element is bound to the first binding site, the method comprising:
 i) optimizing the linker length as a function of the concentration of the first and second binding sites, the size of the activity element and targeting element, the minimum distance between the binding sites of the targeting element and the activity element, and the affinities of the activity element and the targeting element for the first and second binding sites; and   ii) preparing a chimeric activator having an optimal linker length.   
     
     
         40 . A method for manufacturing a chimeric activator comprising an activity element and a targeting element joined by a linker, wherein the targeting element binds to a first binding site on a target cell, and wherein the activity element binds to a second binding site on a target cell, and wherein the linker length has been optimized for maximizing binding efficiency of the activity element to the second binding site when the targeting element is bound to the first binding site, the method comprising:
 i) mutating the activity element such that the binding affinity of the activity element for the second binding site is lower than the binding affinity of the targeting element for the first binding site;   i) optimizing the linker length as a function of the concentration of the first and second binding sites, the size of the activity element and targeting element, the minimum distance between the binding sites of the targeting element and the activity element, and the affinities of the activity element and the targeting element for the first and second binding sites; and   iii) preparing a chimeric activator.   
     
     
         41 . A method for targeted cellular regulation, the method comprising:
 administering to a subject a biologically effective amount of a chimeric activator comprising an activity element and a targeting element joined by a linker, wherein the targeting element binds to a first binding site on a target cell, and wherein the activity element binds to a second binding site on a target cell, and wherein the linker length has been optimized for maximizing binding efficiency of the activity element to the second binding site when the targeting element is bound to the first binding site.   
     
     
         42 . A method for treating cancer, the method comprising:
 administering to a subject having a cancer a therapeutically effective amount of a composition comprising a chimeric activator comprising an activity element and a targeting element joined by a linker, wherein the targeting element binds to a first binding site on a target cell, and wherein the activity element binds to a second binding site on a target cell, and wherein the linker length has been optimized for maximizing binding efficiency of the activity element to the second binding site when the targeting element is bound to the first binding site.