Delivery system for targeted delivery of a therapeutically active payload

Abstract

The present invention provides the modular design and assembly of novel targeting bio-conjugates, exclusively assembled by means of biotin-biotin binding element conjugation, comprising mono-biotinylated cell binding component, a tetrameric biotin-binding element, and mono-biotinylated payload for therapeutic and diagnostic purposes. In addition, there is provided a method of delivering the payload, such as therapeutic oligonucleotides, via mono-biotinylated targeting devices, such as antibodies or ligands, into eukaryotic cells by means of receptor-mediated endocytosis. The targeting bio-conjugates are suitable for use in the areas of medicine, pharmacy and biomedical research.

I claim: 
     
         1 . A delivery system for targeted delivery of a therapeutically active payload, comprising
 an avidin core, wherein the avidin core consists of avidin, neutravidin or streptavidin;   at least one targeting molecule selected from the group consisting of antibody single-chain variable fragments (scFv) and ap tamers,   at least one the therapeutically active payload selected from the group consisting of proteins, peptides and therapeutically active nucleic acids,   
       wherein
 said at least one targeting molecule and said at least one therapeutically active payload are bound to the avidin core; 
 said antibody single-chain variable fragment is comprised in a construct, having the structure:
 antibody single-chain variable fragment—BAP; or 
 antibody single-chain variable fragment—linker—BAP; and 
 
 this construct is mono-biotinylated at the BAP. 
 
     
     
         2 . The delivery system according to  claim 1 , comprising
 a. one antibody single-chain variable fragment and three therapeutically active nucleic acids, or   b. two antibody single-chain variable fragments and two therapeutically active nucleic acids, or   c. three antibody single-chain variable fragments and one therapeutically active nucleic acid.   
     
     
         3 . The delivery system according to  claim 1 , wherein the antibody single-chain variable fragment is selected from scFv(AM1) (SEQ ID NO: 1), scFv(h-AM-1) (SEQ ID NO: 2) and scFv(MR1.1) (SEQ ID NO: 3 or SEQ ID NO: 4). 
     
     
         4 . The delivery system according to  claim 1 , wherein said BAP is selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID NO. 5) 
                 
                   MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPAAGGA 
                 
                   GAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEIN 
                 
                   APTDGKVEKVLVKERDAVQGGQGLIKIGDLEL; 
                 
                     
                 
                   (SEQ ID NO: 6) 
                 
                   VLRSPMPGVVVAVSVKPGDAVAEGQEICVIEAMKMQNSMTAGKTGT 
                 
                   VKSVHCQAGDTVGEGDLLVELE; 
                 
                     
                 
                   (SEQ ID NO: 7) 
                 
                   LX1X2IFEAQKIEWR, wherein 
                 
                   X 1  = any amino acid; and 
                 
                   X 2  = is any amino acid except L, V, I, W, F or Y; 
                 
                     
                 
                   (SEQ ID NO. 8) 
                 
                   GLNDIFEAQKIEWHE; 
                 
                     
                 
                   (SEQ ID NO: 9) 
                 
                   ALNDIFEAQKIEWHA; 
                 
                     
                 
                   (SEQ ID NO. 10) 
                 
                   MAGGLNDIFEAQKIEWHEDTGGS; 
                 
                     
                 
                   (SEQ ID NO: 11) 
                 
                   MSGLNDIFEAQKIEWHEGAPSSR; 
                 
                   and 
                 
                     
                 
                   (SEQ ID NO: 12) 
                 
                   LHHILDAQKMVWNHR. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         5 . The delivery system according to  claim 1 , wherein said linker peptide is selected from the group consisting of
 two amino acids, such as GS;   6 amino acids; and   amino acids, such as the c-myc tag having the amino acid sequence of EQKLISEEDL (SEQ ID NO: 13).   
     
     
         6 . The delivery system according to  claim 1 , wherein said therapeutically active payload is a therapeutically active nucleic acid selected from the group consisting of CpG oligonucleotides, ssDNA, dsDNA, ssRNA or dsRNA, preferably a dsRNA. 
     
     
         7 . The delivery system according to  claim 1 , wherein said therapeutically active payload, preferably the therapeutically active nucleic acid, is biotinylated. 
     
     
         8 . The delivery system according to  claim 1 , wherein said therapeutically active nucleic acid is a siRNA, wherein said siRNA is comprised in a carrier, which comprises a glycodendrimer. 
     
     
         9 . The delivery system according to  claim 8 , wherein said glycodendrimer is a transfection disabled nucleotide carrier, wherein said glycodendrimer comprising a maltose—poly-propylene-imine (mal-PPI) dendrimer comprising one biotin molecule when complexed with siRNA, suitably based on mal19-PPI. 
     
     
         10 . The delivery system according to  claim 1 , wherein said delivery system binds, through the at least one antibody single chain fragment, to a surface antigen, which is specifically expressed at or in cell membranes of cancer cells. 
     
     
         11 . A process for the assembly of a delivery system according to  claim 1  comprising the steps of:
 a) preparing scFv-BAP-biotin conjugates, 
 b) incubating the scFv-BAP-biotin conjugates with the avidin core consisting of avidin, neutravidin or streptavidin, wherein scFv-BAP—avidin or scFv-BAP—neutravidin or scFv-BAP—streptavidin complexes are formed; and 
 c) adding therapeutically active nucleic acid—biotin conjugates and incubating the scFv-BAP—avidin or scFv-BAP—neutravidin or scFv-BAP—streptavidin complexes with the biotinylated therapeutically active nucleic acids; and 
 d) formation of the delivery system by binding of the biotinylated therapeutically active nucleic acids to the avidin, neutravidin or streptavidin of the scFv-BAP—avidin or scFv-BAP—neutravidin or scFv-BAP—streptavidin complexes. 
 
     
     
         12 . The process according to  claim 11 , wherein the therapeutically active component is a siRNA and said siRNA is conjugated to the preformed scFv-BAP—avidin/neutravidin/streptavidin complexes comprising the steps of
 a) preparing a maltose-PPI-biotin conjugate; 
 b) incubating the maltose-PPI-biotin conjugate with the scFv-BAP—avidin or scFv-BAP—neutravidin or scFv-BAP—streptavidin complexes, wherein the maltose-PPI has a cationic charge; 
 c) binding the maltose-PPI-biotin complexes to the avidin, neutravidin or streptavidin of the scFv-BAP—avidin or scFv-BAP—neutravidin or scFv-BAP—streptavidin complexes; 
 d) separately, incubating maltose-PPI with siRNA, wherein maltose-PPI-siRNA dendrimers are formed, which have a weak anionic charge, 
 e) incubating the complexes resulting from step c) with the maltose-PPI-siRNA dendrimers of step d), and 
 f) formation of tumor-targeting polyplexes, comprising scFv-BAP, maltose-PPI-biotin/maltose-PPI-siPvNA dendrimers, which are bound to the avidin/neutravidin core. 
 
     
     
         13 . A pharmaceutical composition comprising the delivery system according to  claim 1 , wherein said pharmaceutical composition optionally further comprises an EGF receptor inhibitor selected from
 tyrosine kinase inhibitors or monoclonal antibodies;   gefitinib, erlotinib, afatinib and osimertinib and cetuximab; and   CimaVax-EGF.   
     
     
         14 . A method for treatment of metabolic diseases, such as familial hypercholesterolemia, viral infections, and proliferative diseases, such as primary tumors like glioblastoma multiforme (GBM) or metastatic cancers, the method comprising:
 administering to a subject in need thereof a therapeutically effective amount of the delivery system according to  claim 1 .   
     
     
         15 . A method for treatment of metabolic diseases, such as familial hypercholesterolemia, viral infections, and proliferative diseases, such as primary tumors like glioblastoma multiforme (GBM) or metastatic cancers, the method comprising:
 administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the delivery system according to  claim 1 .