Chemically activated nanocapsid functionalized for cancer targeting

Abstract

Modified capsid proteins containing at least a portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) having one or more cysteine residues in a surface variable loop or the C-terminus of HEV ORF2, or a portion thereof, are provided. The modified capsid proteins can be used to form hepatitis E virus (HEV) virus like particles (VLPs) having cysteine functional groups exposed on the outer-surface. The exposed cysteine functional groups can be modified via their thiol reactive group. For example, a bioactive agent, such as a cell-targeting ligand, can be conjugated to the one or more cysteines for targeted delivery of chemically activated nanocapsids.

What is claimed is: 
     
         1 . A modified capsid protein comprising a portion of hepatitis E virus (HEV) open Reading Frame 2 (ORF2) protein that is able to form an acid and proteolytically stable HEV virus like particle (VLP), wherein:
 the portion of HEV ORF2 comprises a P-domain of the HEV ORF2 protein;   the P-domain comprises at least one surface variable loop and a C-terminus;   the P-domain comprises a cysteine in the at least one surface variable loop or at the C-terminus; and   the HEV ORF2 portion retains its ability to form an acid and proteolytically stable HEV VLP when the surface variable loop or C-terminal cysteine is chemically derivatized.   
     
     
         2 . The modified capsid protein of  claim 1 , wherein the HEV ORF2 portion retains its ability to form an acid and proteolytically stable HEV VLP when the surface variable loop or C-terminal cysteine is alkylated, agitated, arylated, succinylated, oxidized, or conjugated to a detectable label or bioactive agent. 
     
     
         3 . The modified capsid protein of  claim 1  or  2 , wherein the modified capsid protein comprises an amino acid sequence at least 90%, 95%, or 99% identical, or identical, to residues 112-608 of the HEV ORF2 protein of SEQ ID NO:1, 2, 3, 4, 5, or 6. 
     
     
         4 . The modified capsid protein of  claim 1  or  2 , wherein the at least one P-domain surface variable loop or C-terminal cysteine is conjugated to a detectable label. 
     
     
         5 . The modified capsid protein of  claim 4 , wherein the detectable label comprises a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomic number greater than 20. 
     
     
         6 . The modified capsid protein of  claim 5 , wherein the cluster of elements of group 3 through 18 having an atomic number greater than 20 comprises a gold nanocluster. 
     
     
         7 . The modified capsid protein of  claim 1  or  2 , wherein the at least one P-domain surface variable loop or C-terminal cysteine is conjugated to a bioactive agent. 
     
     
         8 . The modified capsid protein of  claim 7 , wherein the bioactive agent is a heterologous peptide. 
     
     
         9 . The modified capsid protein of  claim 8 , wherein the heterologous peptide is a cell targeting ligand. 
     
     
         10 . The modified capsid protein of  claim 9 , wherein the cell targeting ligand is a cancer cell targeting ligand. 
     
     
         11 . The modified capsid protein of  claim 10 , wherein the cancer cell targeting ligand is LXY30. 
     
     
         12 . The modified capsid protein of  claim 10 , wherein the cancer cell targeting ligand is an antibody that binds an antigen expressed on the surface of a cancer cell. 
     
     
         13 . The modified capsid protein of  claim 9  or  10 , wherein the modified capsid protein further comprises a second cysteine in a P-domain surface variable loop or at the C-terminus of the P-domain. 
     
     
         14 . The modified capsid protein of  claim 13 , wherein the second cysteine is conjugated to a chemotherapeutic. 
     
     
         15 . The modified capsid protein of  claim 13 , wherein the second cysteine is conjugated to a detectable label. 
     
     
         16 . The modified capsid protein of  claim 15 , wherein the detectable label conjugated to the second cysteine comprises a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomic number greater than 20. 
     
     
         17 . The modified capsid protein of  claim 16 , wherein the detectable label conjugated to the second cysteine comprising the cluster of elements of group 3 through 18 having an atomic number greater than 20 comprises a gold nanocluster. 
     
     
         18 . The modified capsid protein of  claim 1  or  2 , wherein the at least one P-domain surface variable loop cysteine is alkylated, acylated, arylated, succinylated, or oxidized. 
     
     
         19 . The modified capsid protein of  claim 1  or  2 , wherein the at least one P-domain surface variable loop cysteine of HEV ORF2 replaces Y485, T489, S533, N573, or T586 of HEV ORF2 or the C-terminal cysteine replaces residue 608 of HEV ORF2. 
     
     
         20 . The modified capsid protein of any one of the previous claims, wherein the modified capsid protein is a component of an acid and proteolytically stable VLP. 
     
     
         21 . The modified capsid protein of  claim 20 , wherein the acid and proteolytically stable HEV VLP encapsulates a bioactive agent. 
     
     
         22 . The modified capsid protein of  claim 21 , wherein the encapsulated bioactive agent is a heterologous nucleic acid, a heterologous peptide, a detectable label, a non-proteinogenic amino acid, an oligosaccharide, a synthetic macromolecule, or a chemotherapeutic. 
     
     
         23 . The modified capsid protein of  claim 22 , wherein the encapsulated detectable label comprises a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomic number greater than 20. 
     
     
         24 . The modified capsid protein of  claim 23 , wherein the encapsulated detectable label comprising the cluster of elements of group 3 through 18 having an atomic number greater than 20 comprises a gold nanocluster. 
     
     
         25 . A composition comprising the modified capsid protein of an one of the previous claims and a pharmaceutically acceptable excipient. 
     
     
         26 . The composition of  claim 25 , wherein the composition comprises an HEV VLP having at least one cygteine within an HEV ORF2 P-domain surface variable loop or C-terminus that is chemically conjugated to a cell targeting ligand, a bioactive agent, or a detectable label. 
     
     
         27 . A nucleic acid comprising a polynucleotide sequence encoding the modified capsid protein of any one of  claims 1 - 24 . 
     
     
         28 . An expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding the modified capsid protein of any one of  claims 1 - 24 . 
     
     
         29 . A cell comprising the nucleic acid of  claim 27  or the expression cassette of  claim 28 . 
     
     
         30 . A cell comprising the modified capsid protein of any one of  claims 1 - 24 . 
     
     
         31 . An organism comprising the modified capsid protein of any one of  claims 1 - 24 . 
     
     
         32 . A method of producing a modified capsid protein comprising cultivating the cell of  claim 29  under conditions suitable to permit expression of the modified capsid protein. 
     
     
         33 . The method of  claim 32  further comprising purifying the capsid protein. 
     
     
         34 . The method of  claim 33  further comprising derivatizing the at least one P-domain surface variable loop or C-terminal cysteine. 
     
     
         35 . The method of  claim 34 , wherein the derivatizing comprises acylating, alkylating, arylating, succinylating, or oxidizing the P-domain surface variable loop or C-terminal cysteine. 
     
     
         36 . The method of  claim 34 , wherein the derivatizing comprises conjugating a bioactive agent to the at least one P-domain surface variable loop or C-terminal cysteine. 
     
     
         37 . A method of directing an HEV VLP to a target cell comprising contacting a cell with the HEV-VLP, wherein the HEV VLP comprises any one of the modified capsid proteins of  claims 1 - 24 , wherein the HEV VLP further comprises a cell targeting moiety having affinity for the target cell conjugated to the at least one P-domain surface variable loop or C-terminal cysteine. 
     
     
         38 . The method of  claim 37 , wherein the HEV VLP further comprises a detectable label, and the method further comprises detecting the detectable label. 
     
     
         39 . The method of  claim 38 , wherein the detecting the detectable label comprises detecting a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomic number greater than 20. 
     
     
         40 . The method of  claim 39 , wherein the detecting the cluster of elements of group 3 through 18 having an atomic number greater than 20 comprises detecting a gold nanocluster. 
     
     
         41 . The method of  claim 37 , wherein the cell targeting moiety having affinity for the target cell conjugated to the at least one P-domain surface variable loop or C-terminal cysteine is a heterologous peptide comprising a cancer cell targeting ligand, and the target cell is a cancer cell. 
     
     
         42 . The method of  claim 41 , wherein the HEV VLP or a component thereof enters the cancer cell. 
     
     
         43 . The method of  claim 37 , wherein the HEV VLP encapsulates a bioactive agent, and the method comprises delivering the bioactive agent to the intracellular region of the cell. 
     
     
         44 . The method of  claim 43 , wherein the encapsulated bioactive agent is a heterologous nucleic acid, a heterologous peptide, a detectable label, a non-proteinogenic amino acid, an oligosaccharide, a synthetic macromolecule, or a chemotherapeutic. 
     
     
         45 . The method of  claim 44 , wherein the encapsulated detectable label comprises a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomic number greater than 20. 
     
     
         46 . The method of  claim 45 , wherein the encapsulated cluster of elements of group 3 through 18 having an atomic number greater than 20 comprises an encapsulated gold nanocluster.