Phosphorylated akt-specific capture agents, compositions, and methods of using and making

Abstract

The present application provides stable peptide-based Akt capture agents and methods of use as detection and diagnosis agents and in the treatment of diseases and disorders. The application further provides methods of manufacturing Akt capture agents using iterative on-bead in situ click chemistry.

1 . A stable, synthetic capture agent that specifically binds Akt, wherein the capture agent comprises a designed anchor ligand, a designed secondary ligand, and optionally, a designed tertiary ligand, wherein the ligands specifically bind phosphorylated Akt. 
     
     
         2 . The capture agent of  claim 1 , wherein the phosphorylated Akt is Akt2. 
     
     
         3 . The capture agent of  claim 1 , wherein the phosphorylated Akt2 is phosphorylated at serine 474. 
     
     
         4 . The capture agent of  claim 1 , wherein the anchor ligand comprises an amino acid sequence wkvk. 
     
     
         5 . The capture agent of  claim 1 , wherein the anchor ligand comprises an amino acid sequence wkvkl. 
     
     
         6 - 12 . (canceled) 
     
     
         13 . The capture agent of  claim 1 , wherein the anchor ligand and secondary ligand are linked together via a 1,4-substituted-1,2,3-triazole residue (Tz4) or via a 1,5-substituted-1,2,3-triazole residue (Tz5). 
     
     
         14 . The capture agent of  claim 1 , wherein the tertiary ligand is covalently bound to the secondary ligand. 
     
     
         15 . The capture agent of  claim 1 , wherein the tertiary ligand is covalently bound to the anchor ligand. 
     
     
         16 . The capture agent of  claim 1 , wherein the capture agent is labeled with a label selected from the group consisting of biotin, copper-DOTA, biotin-PEG3, aminooxyacetate, 19FB, 18FB, 5-Carboxyfluorescein, and FITC-PEG3. 
     
     
         17 . The capture agent of  claim 1 , wherein the capture agent is labeled with the detectable moiety consisting of 64Cu DOTA, 68Ga DOTA, 68Ga NOTA, 18F, A118F NOTA, 64Cu 6sGa sgzr 1241 s6y 94mTc 11omln 11c and 76Br 
     
     
         18 . The capture agent of  claim 1 , wherein the capture agent further comprises a cell penetrating peptide. 
     
     
         19 - 22 . (canceled) 
     
     
         23 . A composition comprising two or more capture agents of  claim 1 . 
     
     
         24 . A method for detecting phosphorylated Akt2 in a biological sample, comprising the step of contacting the biological sample with one or more capture agents of  claim 1 . 
     
     
         25 . (canceled) 
     
     
         26 . A method for detecting phosphorylated Akt2 in a biological sample using an immunoassay, wherein the immunoassay utilizes a capture agent of  claim 1 . 
     
     
         27 . A method of diagnosing a cancer associated with increased Akt2 expression in a subject, the method comprising the steps of:
 a) contacting a biological sample from the subject with one or more capture agents of  claim 1 , wherein each capture agent is linked to a detectable moiety;   b) binding antibody in the biological sample to a substrate; and   c) detecting the moiety linked to the capture agent on the substrate;   wherein detection of the moiety on the substrate diagnoses cancer associated with increased Akt2 expression in the subject.   
     
     
         28 - 29 . (canceled) 
     
     
         30 . A method of monitoring treatment of a cancer associated with increased Akt2 expression in a subject, comprising the steps of:
 a) contacting a first biological sample from the subject with one or more capture agents of  claim 1 , wherein each capture agent is linked to a detectable moiety;   b) detecting the moiety linked to the capture agent, wherein the capture agent is bound to Akt2;   c) administering a treatment for the cancer associated with increased Akt2 expression to the subject;   d) contacting a second biological sample from the subject one or more capture agents of  claim 1 , wherein each capture agent is linked to a detectable moiety; and   e) detecting the moiety linked to the capture agent, wherein the capture agent is bound to Akt2;   (f) comparing the level of moiety detected in step (b) with the level of moiety detected in step (d);   wherein, if less of the moiety is detected in step (e) than in step (b), the treatment is improving cancer in the subject.   
     
     
         31 - 32 . (canceled) 
     
     
         33 . A method of detecting Akt2 in a biological sample, comprising the steps of:
 a) contacting the sample with a capture agent of  claim 1 , wherein the capture agent is linked to a detectable moiety; and   b) detecting the moiety linked to the capture agent, wherein the capture agent is bound to Akt2; and   wherein detection of the moiety indicates the presence of Akt2 in the subject.   
     
     
         34 - 35 . (canceled) 
     
     
         36 . A multiplex capture agent comprising two or more capture agents of  claim 1 , wherein the multiplex capture agent specifically binds Akt2. 
     
     
         37 . The capture agent of  claim 1 , further comprising a tag that encodes a cellular signal to control the Akt protein. 
     
     
         38 - 42 . (canceled) 
     
     
         43 . A method of treating a cancer associated with increased Akt2 expression and/or activity in a subject in need thereof, comprising administering a therapeutically effective amount of a capture agent of  claim 1 . 
     
     
         44 - 45 . (canceled) 
     
     
         46 . A method of inhibiting Akt2 activity in a subject comprising administering to the subject a capture agent of  claim 1 . 
     
     
         47 - 48 . (canceled) 
     
     
         49 . A method of imaging a cancer associated with increased Akt2 expression and/or activity in a subject in need thereof, comprising administering an effective amount of a capture agent of  claim 1 . 
     
     
         50 - 51 . (canceled)