Cell penetrating protein-antibody conjugates and methods of use

Abstract

Provided herein are cell penetrating conjugates. The conjugates include non-cell penetrating proteins connected through a phosphorothioate nucleic acid, wherein the phosphorothioate nucleic acid enhances intracellular delivery of the non-cell penetrating proteins. Also provided are methods and kits including the conjugates provided herein.

1 .- 59 . (canceled) 
     
     
         60 . A cell penetrating conjugate comprising a phosphorothioate nucleic acid connecting a first non-cell penetrating IgG antibody to a second non-cell penetrating IgG antibody, wherein said phosphorothioate nucleic acid enhances intracellular delivery of said first non-cell penetrating IgG antibody and said second non-cell penetrating IgG antibody, and wherein said first non-cell penetrating IgG antibody and said second non-cell penetrating IgG antibody bind an intracellular target. 
     
     
         61 . The cell penetrating conjugate of  claim 60 , wherein said phosphorothioate nucleic acid comprises a first phosphorothioate nucleic acid hybridized to a second phosphorothioate nucleic acid, said first phosphorothioate nucleic acid attached to said first non-cell penetrating IgG antibody and said second phosphorothioate nucleic acid attached to said second non-cell penetrating IgG antibody. 
     
     
         62 . The cell penetrating conjugate of  claim 60 , wherein said phosphorothioate nucleic acid is independently attached to a lysine, arginine, cysteine, or histidine of said first non-cell penetrating IgG antibody. 
     
     
         63 . The cell penetrating conjugate of  claim 62 , wherein said phosphorothioate nucleic acid is attached to a cysteine of said first non-cell penetrating IgG antibody. 
     
     
         64 . The cell penetrating conjugate of  claim 62 , wherein said phosphorothioate nucleic acid is independently attached to a lysine, arginine, cysteine, or histidine of said second non-cell penetrating IgG antibody. 
     
     
         65 . The cell penetrating conjugate of  claim 64 , wherein said phosphorothioate nucleic acid is attached to a cysteine of said second non-cell penetrating IgG antibody. 
     
     
         66 . The cell penetrating conjugate of  claim 60 , wherein said phosphorothioate nucleic acid is attached to said first non-cell penetrating IgG antibody through a first linker and to said second non-cell penetrating IgG antibody through a second linker, and wherein said first linker and said second linker are independently a non-covalent linker. 
     
     
         67 . The cell-penetrating conjugate of  claim 60 , wherein said phosphorothioate nucleic acid is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid residues in length. 
     
     
         68 . The cell-penetrating conjugate of  claim 67 , wherein said phosphorothioate nucleic acid is from about 5 to about 50 nucleic acid residues in length. 
     
     
         69 . The cell penetrating conjugate of  claim 60 , wherein said first non-cell penetrating IgG antibody and said second non-cell penetrating IgG antibody independently have a molecular weight from about 25 kD to about 750 kD. 
     
     
         70 . The cell penetrating conjugate of  claim 60 , wherein said first non-cell penetrating IgG antibody binds a different intracellular target relative to said second non-cell penetrating IgG antibody. 
     
     
         71 . The cell penetrating conjugate of  claim 60 , wherein said first non-cell penetrating IgG antibody binds a different epitope on the intracellular target relative to said second non-cell penetrating IgG antibody. 
     
     
         72 . The cell penetrating conjugate of  claim 60 , wherein said intracellular target is a target of a disease selected from the group consisting of autoimmune disease, inflammatory disease, metabolic disorder, developmental disorder, cardiovascular disease, liver disease, intestinal disease, infectious disease, endocrine disease, neurological disorder, and cancer. 
     
     
         73 . The cell penetrating conjugate of  claim 60 , wherein said intracellular target is an oncogenic transcription factor, an oncoprotein, an immunomodulatory molecule, a DNA binding protein, an epigenetically modified DNA, a signaling molecule or a transcription factor. 
     
     
         74 . The cell penetrating conjugate of  claim 73 , wherein said oncogenic transcription factor is STAT3, Myc, NFkB, API, HIF, or mutant p53. 
     
     
         75 . The cell penetrating conjugate of  claim 73 , wherein said oncoprotein is Ras, Raf, MAPK, PI3 kinase, AKT, BTK, JAKs, or SRC. 
     
     
         76 . The cell penetrating conjugate of  claim 73 , wherein said immunomodulatory molecule is FOXp3, T-BET, GAT A3, STAT1, 2, 3, 4, 5, or 6. 
     
     
         77 . The cell penetrating conjugate of  claim 60 , wherein said intracellular target is a cancer target selected from the group consisting of STAT3, NFKB, PKB/Akt, Myc, cyclin Dl, receptor tyrosine kinases (RTKs), EGFR, VEGFR, PDGFR, Src, Ras, Abl, BCR-Abl, NPM-Alk, Janus kinases (JAKs), Brutun's tyrosine kinase (BTK), and viral oncoproteins. 
     
     
         78 . The cell penetrating conjugate of  claim 60 , wherein said intracellular target is a viral protein or viral transcript of a human immunodeficiency virus (HIV), influenza virus, herpes simplex cirus, epstein barr virus, cytomegalovirus, human papilloma virus, or hepatitis virus. 
     
     
         79 . A cell comprising the cell penetrating conjugate of  claim 60 . 
     
     
         80 . A pharmaceutical composition comprising the cell penetrating conjugate of claim  1  and a pharmaceutically acceptable carrier. 
     
     
         81 . A method of delivering a non-cell penetrating IgG antibody into a cell comprising contacting the cell with said cell penetrating conjugate of  claim 60 , wherein said first non-cell penetrating IgG antibody binds a nuclear protein in the cytoplasm, thereby forming a first non-cell penetrating antibody-nuclear protein complex, and said second non-cell penetrating IgG antibody binds a nuclear protein in the cytoplasm, thereby forming a second non-cell penetrating antibody-nuclear protein complex. 
     
     
         82 . A method of treating a disease selected from the group consisting of autoimmune disease, developmental disorder, inflammatory disease, metabolic disorder, cardiovascular disease, liver disease, intestinal disease, infectious disease, endocrine disease, neurological disorder, and cancer in a subject in need thereof, said method comprising administering to the subject an effective amount of the cell penetrating conjugate of  claim 60 , thereby treating the disease in said subject.