Methods and compositions for 18f-radiolabeling of biologics

Abstract

The invention relates to water soluble 18F-prosthetic groups and the synthesis and use of 18F-labeled biological molecules containing the 18F-prosthetic groups for imaging various processes within the body, for detecting the location of molecules associated with disease pathology, and for monitoring disease progression are disclosed.

1 - 55 . (canceled) 
     
     
         56 . A method of obtaining an image of an  18 F-radiolabeled protein-based probe comprising  18 F-radiolabeled prosthetic group, a bifunctional conjugating (BFC) moiety and a protein with the following structure, 
       
         
           
           
               
               
           
         
       
       wherein the  18 F is ortho to the N atom, x is an integer from 1 to 8, and the BFC comprises a cyclooctyne moiety and a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl or thiol functional group on the protein, or pharmaceutically acceptable salt thereof, the method comprising,
 a) administering the  18 F-radiolabeled protein-based probe to a subject; and 
 b) imaging in vivo the distribution of the  18 F-radiolabeled protein-based probe by positron emission tomography (PET) scanning. 
 
     
     
         57 . (canceled) 
     
     
         58 . The method of  claim 56 , 
       wherein the presence and location of the  18 F-radiolabeled protein-based probe above background is indicative of the presence and location of the disease. 
     
     
         59 . The method of  claim 58 ,
 comprising administering to the subject the  18 F-radiolabeled protein-based probe at one or more time points and obtaining an image of at least a portion of the subject at each time point; wherein the dimension and location of the diseased cells or tissue at each time point is indicative of the progress of the disease.   
     
     
         60 . The method of  claim 58 ,
 wherein the level and distribution of the radioactive emissions in the diseased cells or tissues is a quantitative measure of the diseased cells or tissues.   
     
     
         61 . (canceled) 
     
     
         62 . A method of obtaining a quantitative image of tissues or cells expressing a target protein, the method comprising contacting the cells or tissue with an  18 F-radiolabeled protein-based probe of which binds to the target protein, and detecting or quantifying the tissue expressing the target protein using positron emission tomography (PET), wherein the  18 F-radiolabeled protein-based probe comprises  18 F-radiolabeled prosthetic group, a bifunctional conjugating (BFC) moiety and a protein with the following structure, 
       
         
           
           
               
               
           
         
       
       wherein the  18 F is ortho to the N atom, x is an integer from 1 to 8, and the BFC comprises a cyclooctyne moiety and a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl or thiol functional group on the protein, or pharmaceutically acceptable salt thereof. 
     
     
         63 . A method of screening for an agent for treating a disease comprising the steps of
 (a) contacting cells expressing a target protein associated with the disease with an  18 F-radiolabeled protein-based probe which binds to the target protein in the presence and absence of a candidate agent; and   (b) imaging the cells in the presence and absence of the candidate agent using positron emission tomography (PET),   
       wherein a decrease in the amount of radioactive emissions in the presence of the candidate agent is indicative of that the agent binds to the target protein, and 
       wherein the  18 F-radiolabeled protein-based probe comprises  18 F-radiolabeled prosthetic group, a bifunctional conjugating (BFC) moiety and a protein with the following structure, 
       
         
           
           
               
               
           
         
       
       wherein the  18 F is ortho to the N atom, x is an integer from 1 to 8, and the BFC comprises a cyclooctyne moiety and a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl or thiol functional group on the protein, or pharmaceutically acceptable salt thereof. 
     
     
         64 - 70 . (canceled) 
     
     
         71 . A kit comprising the reaction precursors for use in producing the  18 F-radiolabeled protein-based probe comprising a  18 F-radiolabeled prosthetic group, a bifunctional conjugating (BFC) moiety and a protein with the following structure, 
       
         
           
           
               
               
           
         
       
       wherein the  18 F is ortho to the N atom, x is an integer from 1 to 8, and the BFC comprises a cyclooctyne moiety and a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl or thiol functional group on the protein, or pharmaceutically acceptable salt thereof, and instructions for producing the  18 F-radiolabeled protein-based probe. 
     
     
         72 . A method for preparing an  18 F-radiolabeled compound with the following structure, 
       
         
           
           
               
               
           
         
       
       wherein x is an integer from 1 to 8, the method comprising the steps of
 (a) providing a solution of a compound a with the following structure: 
 
       a 
       
         
           
           
               
               
           
         
       
       wherein x is an integer from 1 to 8, and R is NO 2 , Br, F or 
       
         
           
           
               
               
           
         
       
       and is ortho to the N atom of the pyridine ring;
 (b) providing a mixture of  18 F in  18 O water, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and a weak base; 
 c) drying the mixture from step b) to form a solid; and 
 d) reacting the solution from step a) with the solid from step c) to form the  18 F-labeled compound. 
 
     
     
         73 - 81 . (canceled) 
     
     
         82 . The method of  claim 72 , wherein the  18 F-labeled compound has the structure 
       
         
           
           
               
               
           
         
       
       wherein the method comprises
 a) providing a solution of the compound of the structure 
 
       
         
           
           
               
               
           
         
       
       where X is NO 2 , Br or 
       
         
           
           
               
               
           
         
         b) providing a mixture of  18 F in  18 O water, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and weak base; 
         c) drying the mixture from step b) to form a solid; and 
         d) reacting the solution from step a) with the solid from step c) to form the  18 F-labeled compound. 
       
     
     
         83 . (canceled) 
     
     
         84 . A method of producing an  18 F-radiolabeled protein-based probe comprising an  18 F-radiolabeled prosthetic group, a bifunctional conjugating (BFC) moiety and a protein with the following structure, 
       
         
           
           
               
               
           
         
       
       wherein the  18 F is ortho to the N atom, x is an integer from 1 to 8, and the BFC comprises a cyclooctyne moiety and a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl or thiol functional group on the protein, the method comprising coupling the  18 F-radiolabeled prosthetic group comprising the structure 
       
         
           
           
               
               
           
         
       
       with a protein comprising a bifunctional conjugating (BFC) moiety using a bioorthogonal click reaction. 
     
     
         85 . The method of  claim 84 , wherein the bioorthogonal click reaction is metal free. 
     
     
         86 - 87 . (canceled) 
     
     
         88 . The method of  claim 56 , wherein the  18 F-radiolabeled protein-based probe has the following structure, 
       
         
           
           
               
               
           
         
       
       wherein the maleimide group of the BFC is covalently linked to a thiol group on a cysteine residue of the protein. 
     
     
         89 . The method of  claim 88 , wherein the protein portion of the  18 F-radiolabeled protein-based probe comprises a fibronectin type III (Fn3) domain. 
     
     
         90 . The method of  claim 88 , wherein the protein portion of the  18 F-radiolabeled protein-based probe comprises an antibody or antibody fragment. 
     
     
         91 . The method of  claim 62 , wherein the  18 F-radiolabeled protein-based probe has the following structure, 
       
         
           
           
               
               
           
         
       
       wherein the maleimide group of the BFC is covalently linked to a thiol group on a cysteine residue of the protein. 
     
     
         92 . The method of  claim 91 , wherein the protein portion of the  18 F-radiolabeled protein-based probe comprises a fibronectin type III (Fn3) domain. 
     
     
         93 . The method of  claim 91 , wherein the protein portion of the  18 F-radiolabeled protein-based probe comprises an antibody or antibody fragment. 
     
     
         94 . The method of  claim 63 , wherein the  18 F-radiolabeled protein-based probe has the following structure, 
       
         
           
           
               
               
           
         
       
       wherein the maleimide group of the BFC is covalently linked to a thiol group on a cysteine residue of the protein. 
     
     
         95 . The method of  claim 94 , wherein the protein portion of the  18 F-radiolabeled protein-based probe comprises a fibronectin type III (Fn3) domain. 
     
     
         96 . The method of  claim 94 , wherein the protein portion of the  18 F-radiolabeled protein-based probe comprises an antibody or antibody fragment.