Selective uv crosslinking of peptides and functional moieties to immunoglobulins

Abstract

The invention provides for a method of crosslinking a hetero-bifunctional photo crosslinking compound to an immunoglobulin having at least one heterocyclic photo reactive group and at least one non-photo reactive group where the non-photo reactive group is coupled to an effector molecule and the photo reactive group is coupled to the nucleotide binding site of an immunoglobulin. Alternatively, the photo crosslinker contains an orthogonal reactive group such as a thiol, which can be coupled to an effector molecule or functionalized ligand.

What is claimed is: 
     
         1 . A method of site specific photo crosslinking an immunoglobulin, the method comprising:
 a) providing an immunoglobulin, the immunoglobulin having a conserved nucleotide binding site located away from the antigen binding site of the F V  domain of the immunoglobulin;   b) providing a hetero-bifunctional photo-reactive crosslinker, the hetero-bifunctional photo-reactive crosslinker having at least one photo reactive heterocyclic functional group that interacts with the conserved nucleotide binding site of the immunoglobulin, and at least one non-photo reactive functional group;   c) mixing the immunoglobulin with the hetero-bifunctional photo-reactive crosslinker to provide a mixture; and   d) exposing the mixture to ultra-violet light so that the at least one photo reactive functional group of the hetero-bifunctional photo-reactive crosslinker is covalently coupled within the nucleotide binding site of the immunoglobulin.   
     
     
         2 . The method of  claim 1  wherein the at least one heterocyclic functional group is an indole compound. 
     
     
         3 . The method of  claim 2  wherein the photo reactive functional group is indole-3-butyric acid. 
     
     
         4 . The method of  claim 1  wherein the at least one non-photo reactive functional group is coupled to a surface in an orientation specific manner whereby the antigen binding sites are oriented away from the surface and available for antigen binding such that the immunoglobulin retains about 90%-100% antigen binding activity. 
     
     
         5 . The method of  claim 1  wherein the at least one non-photo reactive functional group is coupled to an effector molecule. 
     
     
         6 . The method of  claim 4  wherein the surface is a drug delivery system or a diagnostic contrast agent. 
     
     
         7 . The method of  claim 6  wherein the drug delivery system comprises a liposome, a micelle, a nanoparticle, a quantum dot or a dendrimer. 
     
     
         8 . The method of  claim 5  where the effector molecule is a labeling molecule, an affinity tag, a chemotherapeutic, a cytotoxic agent, an active peptide, a contrast agent, a radiolabel, DNA, or a small molecule inhibitor. 
     
     
         9 . The method of  claim 8  wherein the effector molecule is biotin, wherein the biotin is accessible to bind to streptavidin, where the streptavidin at least partially coats a surface. 
     
     
         10 . The method of  claim 4  or  9  wherein the surface is a nanoparticle, a bead, a microfluidic device, an ELISA plate, or a microarray device. 
     
     
         11 . The method of  claim 8  wherein the labeling molecule has fluorescent, absorbent, contrast, or radiolabel function. 
     
     
         12 . A method of site specific photo crosslinking of an orthogonally reactive functional group to an immunoglobulin, the method comprising:
 a) providing an immunoglobulin, the immunoglobulin having a conserved nucleotide binding site located away from the antigen binding site of the FA, domain of the immunoglobulin; and   b) providing a hetero-bifunctional crosslinker having at least a first functional group and at least a second functional group where the first functional group is a heterocyclic photo reactive functional group and the second functional group is a thiol functional group;   c) mixing the immunoglobulin with the hetero-bifunctional crosslinker to provide a mixture;   d) exposing the mixture to ultra-violet light so that the first functional group is covalently coupled within the nucleotide binding site of the immunoglobulin; and   e) reacting the thiol functional group with a thiol reactive functionalized ligand;   thereby providing a functionalized immunoglobulin having site specific thiolation.   
     
     
         13 . The method of  claim 12  wherein the heterocyclic photo reactive functional group is an indole compound. 
     
     
         14 . The method of  claim 12  wherein the heterocyclic photo reactive functional group is indole-3-butyric acid. 
     
     
         15 . The method of  claim 12  wherein the thiol functional group is a cysteine residue. 
     
     
         16 . The method of  claim 12  wherein the functionalized ligand is coupled to a thiol-reactive surface. 
     
     
         17 . The method of  claim 12  wherein the thiol functional group is coupled to the functionalized ligand coated surface in an orientation specific manner whereby the antigen binding sites are oriented away from the surface and available for antigen binding such that the immunoglobulin retains about 90% to about 100% antigen binding activity. 
     
     
         18 . The method of  claim 16  wherein the functionalized ligand coated surface is a drug delivery system. 
     
     
         19 . The method of  claim 18  wherein the drug delivery system comprises a liposome, a micelle, a nanoparticle, a quantum dot or a dendrimer. 
     
     
         20 . The method of  claim 12  wherein the functionalized ligand is a labeling molecule, an affinity tag, a chemotherapeutic, a cytotoxic agent, an active peptide, a contrast agent, a radiolabel, DNA, or a small molecule inhibitor. 
     
     
         21 . The method of  claim 20  wherein the affinity tag is biotin, wherein the biotin is bound to streptavidin, where the streptavidin at least partially coats a surface. 
     
     
         22 . The method of  claim 17  or  21  wherein the surface is the surface of a nanoparticle, a bead, a microfluidic device, an ELISA plate, or a microarray device. 
     
     
         23 . The method of  claim 20  wherein the active peptide is selected from the group consisting of cell internalization sequences, receptor targeting sequences and mimitopes. 
     
     
         24 . The method of  claim 20  wherein the labeling molecule has fluorescent, absorbent, contrast, or radiolabel function. 
     
     
         25 . An isolated immunoglobulin-ligand conjugate comprising: an immunoglobulin having a conserved nucleotide binding site located away from the antigen binding site of the FA, domain of the immunoglobulin, the ligand being a hetero-bifunctional crosslinker, the ligand having at least one functional group that is a heterocyclic photo reactive functional group, the ligand also having at least one non-photo reactive functional group, where the at least one heterocyclic photo reactive functional group being coupled to the nucleotide binding site and the at least one non-photo reactive functional group being coupled to an effector molecule.