Pharmaceutical Compositions for High-Capacity Targeted Delivery

Abstract

Provided herein are aptamers and pharmaceutical compositions comprising the same. In some embodiments, the aptamer selectively binds a protein of interest such as an extracellular receptor protein of interest (e.g., a cancer cell extracellular receptor protein, which may be differentially expressed in some embodiments). In some embodiments, the aptamer is directly linked by covalent bonding (e.g., via a geminal diamine linkage) to from 2 to 10 toxin compounds. Also provided herein is a method of selecting an aptamer that specifically binds to a protein expressed by a cell of interest, wherein in some embodiments the aptamer comprises at least one binding site for one or more active compounds. In some embodiments, primer regions flanking the variable region of the aptamers in the pool contains from 1 to 10 mismatches with respect to said forward or reverse primer.

That which is claimed is: 
     
         1 . A pharmaceutical composition comprising an aptamer in a pharmaceutically acceptable carrier,
 wherein said aptamer selectively binds a cancer cell extracellular receptor protein, and   wherein said aptamer is directly linked by covalent bonding to from 2 to 10 toxin compounds.   
     
     
         2 . The composition of  claim 1 , wherein said covalent bonding comprises a geminal diamine linkage. 
     
     
         3 . The composition of  claim 2 , wherein at least one of said toxin compounds is an anthracycline or a taxane having a free amine. 
     
     
         4 . The composition of  claim 3 , wherein said aptamer is directly linked by covalent bonding to said anthracycline or said taxane at a CpG binding site on said aptamer. 
     
     
         5 . The composition of  claim 1 , wherein the half life of said covalent bonding to the toxin compounds is at least 5 hours in human blood plasma. 
     
     
         6 . The composition of  claim 1 , wherein said aptamer comprises at least one FdUMP. 
     
     
         7 . The composition of  claim 1 , wherein said aptamer is provided as a dimeric complex. 
     
     
         8 . The composition of  claim 7 , wherein said cancer cell extracellular receptor protein is a dimeric protein and said dimeric complex comprises a first nucleic acid that selectively binds to said cancer cell extracellular receptor protein, a second nucleic acid that also selectively binds to said cancer cell extracellular receptor protein, and a linker connecting said first and second nucleic acids. 
     
     
         9 . The composition of  claim 8 , wherein said dimeric protein is prostate specific membrane antigen (PSMA), transferrin receptor, carbonic anhydrase XII, or an ErbB receptor. 
     
     
         10 . The composition of  claim 8 , wherein said linker is double-stranded poly-DNA. 
     
     
         11 . The composition of  claim 10 , wherein said double-stranded poly-DNA comprises at least one CpG binding site. 
     
     
         12 . The composition of  claim 10 , wherein said double-stranded poly-DNA comprises a poly-T/poly-A region, wherein some or all of the poly-T residues are replaced by poly-FdU. 
     
     
         13 . A method of selecting an aptamer that specifically binds to an extracellular surface protein expressed by a cell of interest, wherein said aptamer comprises at least one binding site for one or more active compounds, said method comprising the steps of:
 a. combining a first pool comprising different nucleic acids with said extracellular surface protein or a fragment thereof, wherein said nucleic acids comprise:
 i. a variable region from 20 to 60 nucleotides long; and 
 ii. primer regions flanking said variable region and capable of binding a forward and reverse primer during amplification, wherein each of said primer regions contains from 1 to 10 mismatches with respect to said forward or reverse primer; and 
 iii. one or more binding sites for said active compounds; 
   b. selecting a first subpopulation of nucleic acids from said first pool, said first subpopulation comprising at least one nucleic acid that specifically binds to said extracellular surface protein;   c. amplifying said at least one nucleic acid of said first subpopulation, wherein said amplifying is performed under conditions that allow priming at sites other than the primer regions; and   d. selecting a second subpopulation comprising at least one nucleic acid species from said first subpopulation which comprises at least one binding site for one or more active compounds;   to thereby select said aptamer.   
     
     
         14 . The method of  claim 13 , wherein said aptamer has a shorter length than the nucleic acids of said first pool as a result of the priming at sites other than the primer regions. 
     
     
         15 . The method of  claim 13 , wherein said binding sites for said active compounds comprise a CpG site. 
     
     
         16 . The method of  claim 13 , wherein said extracellular surface protein is expressed by a cancer cell. 
     
     
         17 . The method of  claim 13 , wherein said binding sites for said one or more active compounds comprise one or more CpG sites in said primer region. 
     
     
         18 . The method of  claim 13 , wherein said aptamer is capable of being internalized by said cell of interest 
     
     
         19 . The method of  claim 13  further comprising the step of covalently binding said one or more active compounds to said aptamer. 
     
     
         20 . The method of  claim 19 , wherein said active compounds comprise an anthracycline or a taxane. 
     
     
         21 . The method of  claim 20 , wherein said anthracycline or said taxane is released by said aptamer at pH less than or equal to 6. 
     
     
         22 . The method of  claim 21 , wherein formaldehyde is produced when said anthracycline or said taxane is released from said aptamer.