US2007117153A1PendingUtilityA1

Molecular conjugate

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Assignee: BIENIARZ CHRISTOPHERPriority: Nov 23, 2005Filed: Nov 21, 2006Published: May 24, 2007
Est. expiryNov 23, 2025(expired)· nominal 20-yr term from priority
Inventors:Christopher BieniarzJulia Ashworth-SharpeCasey A. KernagJerome W. KosmederMark Lefever
G01N 33/532A61K 47/6801C07C 327/28C12N 9/16C07C 323/60C07C 323/22C12N 9/96A61K 39/44G01N 33/535C12Y 301/03001G01N 33/53C07K 16/00A61K 47/6815G01N 33/581C07C 319/02
56
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Claims

Abstract

A method is disclosed for making a conjugate of two molecules using a hydrazide thiol linker. In a particular working embodiment, an Fc-specific antibody-enzyme conjugate is made using the method and demonstrated to provide exceptional staining sensitivity and specificity in immunohistochemical and in situ hybridization assays.

Claims

exact text as granted — not AI-modified
1 . A method for forming a conjugate of two or more molecules, comprising: 
 reacting a hydrazide thiol linker with a first molecule having a hydrazide-reactive group to form a thiolated first molecule, wherein reacting the hydrazide thiol linker with the first molecule comprises reacting under conditions where a thiol group of the hydrazide thiol linker is substantially present in its neutral acid form; and    reacting the thiolated first molecule with a second molecule, the second molecule having a thiol-reactive group, to form the conjugate.    
   
   
       2 . The method of  claim 1 , wherein the first molecule comprises a specific binding molecule and the second molecule comprises a detectable label.  
   
   
       3 . The method of  claim 1 , wherein reacting under conditions where the thiol group of the hydrazide thiol linker compound is substantially present in its neutral acid form comprises reacting at a pH of less than about 7.  
   
   
       4 . The method of  claim 1 , wherein reacting under conditions where the thiol group of the hydrazide thiol linker compound is substantially present in its neutral acid form comprises reacting from about pH=3 to about pH=7.  
   
   
       5 . The method of  claim 1 , wherein reacting under conditions where the thiol group of the hydrazide thiol linker compound is substantially present in its neutral acid form comprises reacting from about pH=4 to about pH=6.  
   
   
       6 . The method of  claim 1 , wherein the hydrazide-reactive group of the first molecule comprises an aldehyde group.  
   
   
       7 . The method of  claim 6 , wherein the first molecule comprises a glycosylated molecule and the aldehyde group is introduced to the first molecule by oxidation of a glycosylated portion of the first molecule.  
   
   
       8 . The method of  claim 7 , wherein the glycosylated molecule comprises an antibody and the aldehyde group is introduced to an Fc portion of the antibody.  
   
   
       9 . The method of  claim 1 , wherein the thiol-reactive group of the second molecule comprises a maleimide group introduced to the second molecule.  
   
   
       10 . The method of  claim 9 , wherein the maleimide group introduced to the second molecule is introduced using a NHS-PEG-maleimide linker.  
   
   
       11 . The method of  claim 9 , wherein the second molecule comprises a detectable label.  
   
   
       12 . The method of  claim 1 , wherein the hydrazide thiol linker compound is one or more of MBH, MBCH, MAMBH, THMBH, BTAL, BTHL, TAGD, THGD, a PEG-based hydrazide thiol linker, a multifunctional hydrazide thiol linker, a PEG-based multifunctional hydrazide thiol linker, and a polyacrylamide hydrazide linker.  
   
   
       13 . The method of  claim 12 , wherein the hydrazide thiol linker compound is one or more of MBH, MBCH or a mercapto-dPEG-hydrazide linker.  
   
   
       14 . The method of  claim 1 , wherein the hydrazide thiol linker compound has the formula:  
     
       
         
         
             
             
         
       
     
     wherein n=1, 2 or 3; and R 1  is H, —CONHNH 2 , or —CO—A—CONHNH 2 , where A is a divalent group having between 1 and 100 carbon atoms.  
   
   
       15 . The method of  claim 1 , wherein the hydrazide thiol linker compound has the formula:  
     
       
         
         
             
             
         
       
     
     wherein m=2 to 50, R 2  is H, —CONHNH 2 , or —CO—A—CONHNH 2 , where A is a divalent group having between 1 and 100 carbon atoms, and X and Y are independently a bond or a divalent group having 1 to 20 carbons.  
   
   
       16 . A conjugate prepared according to the method of  claim 1 .  
   
   
       17 . A conjugate comprising an antibody covalently bonded to a detectable label through a hydrazide thiol linker, wherein the hydrazide thiol linker is one or more of MBH, MBCH, MAMBH, THMBH, BTAL, BTHL, TAGD, THGD, a PEG-based hydrazide-thiol linker, a multifunctional hydrazide thiol linker, a PEG-based multifunctional hydrazide thiol linker, and a polyacrylamide hydrazide linker, wherein a hydrazide group of the linker is covalently bonded to an Fc region of the antibody.  
   
   
       18 . The conjugate of  claim 17 , wherein the hydrazide thiol linker comprises a PEG-based hydrazide thiol linker.  
   
   
       19 . The conjugate of  claim 18 , wherein the PEG-based hydrazide thiol linker comprises a mercapto-dPEG-hydrazide linker.  
   
   
       20 . The conjugate of  claim 17 , wherein the hydrazide thiol linker comprises MBH or MBCH.  
   
   
       21 . The conjugate of  claim 17 , wherein the detectable label comprises an enzyme, a fluorescent molecule, a hapten or a fluorescent nanoparticle.  
   
   
       22 . The conjugate of  claim 21 , wherein the detectable label comprises an enzyme.  
   
   
       23 . The conjugate of  claim 22 , wherein the detectable label comprises an enzyme selected from the group consisting of alkaline phosphatase and horseradish peroxidase.  
   
   
       24 . The conjugate of  claim 23 , wherein the detectable label comprises alkaline phosphatase.  
   
   
       25 . The conjugate of  claim 24 , wherein the alkaline phosphatase comprises cross-linked alkaline phosphatase.  
   
   
       26 . The conjugate of  claim 17 , wherein the antibody comprises an anti-hapten antibody.  
   
   
       27 . The conjugate of  claim 17 , wherein the antibody comprises and anti-antibody antibody.  
   
   
       28 . A hydrazide thiol linker having the formula:  
     
       
         
         
             
             
         
       
     
     wherein R 1  is H or —CONHNH 2  and n=1, 2 or 3.  
   
   
       29 . A kit comprising a hydrazide thiol linker having the formula:  
     
       
         
         
             
             
         
       
     
     wherein R 1  is H or —CONHNH 2  and n=1, 2 or 3; and 
 instructions for performing the method of  claim 1 .  
 
   
   
       30 . A kit comprising a hydrazide thiol linker having the formula:  
       H 2 N—NH—CO—(CH 2 —CH 2 —O) t —CH 2 —CH 2 —SH  
     where t=2 to 50; and 
 instructions for performing the method of  claim 1.

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