Macromolecular conjugates for visualization and separation of proteins and cells

Abstract

Macromolecular water-soluble conjugates based on synthetic copolymers to which at least one affinity tag, at least one imaging probe, and at least one targeting ligand are bound via covalent bonds. The macromolecular conjugate may be used in identification, visualization, quantification or isolation of proteins and/or cells. The targeting ligand may be attached to the synthetic copolymer via a flexible linker.

1 : Synthetic macromolecular conjugate for selective interaction with proteins, characterized in that it contains a copolymer to which at least one affinity tag, at least one imaging probe and at least one targeting ligand are bound via covalent bonds. 
     
     
         2 : The macromolecular conjugate according to  claim 1 , characterized in that said copolymer is a copolymer obtainable by copolymerization of at least one monomer of Formula 1: 
       
         
           
           
               
               
           
         
         wherein: 
         R 1  is selected from H, CH 3 ; 
         R 2  is selected from NH 2 , NH—CH 2 —CH(OH)—CH 3 , NH—CH 3 , NH—CH 2 CH 3 , NH—CH 2 CH 2 —OH, NH—CH 2 CH 2 CH 2 —OH, NHC(CH 2 OH) 3 , NH—CH 2 CH 2 —N + (CH 3 ) 3 Cl − , O—CH 2 CH 2 —OH, O—(CH 2 CH 2 O) 2 —H O—(CH 2 CH 2 O) 3 —H, O—CH 2 CH 2 —N + (CH 3 ) 3 Cl − , NH—(CH 2 ) 3 N + (CH 3 ) 2 —(CH 2 ) 2 —COO − ; 
         and at least one monomer of Formula 2: 
       
       
         
           
           
               
               
           
         
         wherein: 
         R 1  is selected from H, CH 3 , and 
         X is selected from NH—(CH 2 ) 2 —CO, NH—(CH 2 ) 3 —CO, NH—(CH 2 ) 4 —CO, NH—(CH 2 ) 5 —CO, Gly, GlyGly, GlyPheLeuGly, and 
         R 3  is selected from 
       
       
         
           
           
               
               
           
         
         whereas at least one reactive group R 3  is replaced by the targeting ligand, at least one reactive group R 3  is replaced by the affinity tag, and at least one reactive group R 3  is replaced by the imaging probe. 
       
     
     
         3 : The macromolecular conjugate according to  claim 1 , characterized in that the molecular weight of the conjugate is preferably in the range of 1000 to 500000 g/mol, preferably in the range of 20000 to 150000 g/mol. 
     
     
         4 : The macromolecular conjugate according to  claim 1 , characterized in that the targeting ligand is a moiety capable of selectively binding to the targeted protein or peptide sequence, in particular the targeting ligand is selected from the group comprising an inhibitor or substrate of the targeted enzyme, an agonist or antagonist of the targeted receptor, a ligand of the targeted protein resin. 
     
     
         5 : The macromolecular conjugate according to  claim 1 , characterized in that the targeting ligand may be attached to the synthetic copolymer via a flexible linker, preferably via a flexible linker based on polyethylene glycol, peptide, preferably a peptide having a molecular weight from 100 to 5000 g/mol, or nucleic acid, preferably a nucleic acids comprising 1 to 40 nucleotides, or oligosaccharide, preferably an oligosaccharide containing 1 to 40 monosaccharides. 
     
     
         6 : The macromolecular conjugate according to  claim 1 , characterized in that the affinity tag is selected from biotin, His-tag, FLAG tag, HA tag, Strep-tag, Avi-Tag, GST-tag, c-myc-tag, V5-tag, E-tag, S-tag, SBP-tag, poly(Glu)-tag, calmodulin tag. 
     
     
         7 : The macromolecular conjugate according to  claim 1 , characterized in that the imaging probe is selected from the group comprising fluorescent moieties, radionuclides and metal complexes. 
     
     
         8 : The macromolecular conjugate according to  claim 6 , characterized in that the imaging probe is selected from the group comprising fluorophores with an excitation maximum in the range of 350 to 850 nm, preferably ATTO488 or DY676; lanthanide complexes, preferably of Gd, Mn, Dy, Eu; radionuclide complexes  64 Cu,  68 Ga,  18 F,  99m Tc,  123 I,  125 I,  131 I,  57 Co,  51 Cr,  67 Ga,  64 Cu,  111 In,  90 Y. 
     
     
         9 : Use of the macromolecular conjugate according to  claim 1  for identification, visualization, quantification or isolation of proteins and/or cells in vitro. 
     
     
         10 : Use of the macromolecular conjugate according to  claim 1 , in an immunochemical methods, preferably selected from ELISA, flow cytometry, immunocytochemistry, immunohistochemistry, Wester blotting and modifications thereof. 
     
     
         11 : Use of the macromolecular conjugate according to  claim 1 , for screening of inhibitors, ligands, and compounds and substances capable of binding to the target protein. 
     
     
         12 : The macromolecular conjugate according to  claim 1  for use in identification, visualization, quantification or isolation of proteins and/or cells in vivo, in particular for use in methods of magnetic resonance, radiodiagnostics and radiotherapy. 
     
     
         13 : Use according to any  claim 9 , wherein the target protein is GCPII, GCPIII, CA-II, CA-VII, CA-IX, HIV-1 protease, aspartate protease.