Selective uv crosslinking of peptides and functional moieties to immunoglobulins

Abstract

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.

1 .- 20 . (canceled) 
     
     
         21 . A method of site-specific photo crosslinking an immunoglobulin comprising:
 a) mixing a hetero-bifunctional photo-reactive crosslinker with an immunoglobulin to form a mixture, the hetero-bifunctional photo-reactive crosslinker having at least one photo reactive heterocyclic functional group that interacts with a conserved nucleotide binding site located away from the antigen binding site of the Fv domain of the immunoglobulin, wherein the at least one photo reactive heterocyclic functional group is an indole, and has at least one non-photo reactive functional group; and   b) exposing the mixture to ultra-violet light so that the at least one photo reactive heterocyclic functional group of the hetero-bifunctional photo-reactive crosslinker is covalently coupled within the nucleotide binding site of the immunoglobulin, wherein the concentration of the hetero-bifunctional photo-crosslinker in the mixture is about 100 μm to about 1,600 μm and the ratio of the immunoglobulin to the hetero-bifunctional photo-reactive crosslinker in the mixture is about 1:10 to about 1:80.   
     
     
         22 . The method of  claim 21  wherein the photo reactive heterocyclic functional group is indole-3-butyric acid. 
     
     
         23 . The method of  claim 21  wherein the at least one non-photo reactive functional group is coupled to an effector molecule. 
     
     
         24 . The method of  claim 23  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. 
     
     
         25 . The method of  claim 24  wherein the labeling molecule has fluorescent, absorbent, contrast, or radiolabel function. 
     
     
         26 . The method of  claim 24  wherein the labeling molecule is fluorescein isothiocyanate (FITC) or a derivative thereof. 
     
     
         27 . The method of  claim 24  wherein the active peptide is selected from the group consisting of a cell internalization sequence, a receptor targeting sequence, and a mimitope. 
     
     
         28 . The method of  claim 27  wherein the cell internalization sequence is an Arg-Gly-Asp (RGD) peptide. 
     
     
         29 . The method of  claim 24  wherein the effector molecule is biotin, wherein the biotin is accessible to bind to streptavidin, and the streptavidin at least partially coats a surface. 
     
     
         30 . The method of  claim 29  wherein the surface is a nanoparticle, a bead, a microfluidic device, an ELISA plate, or a microarray device. 
     
     
         31 . The method of  claim 21  wherein after the exposing step, 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. 
     
     
         32 . The method of  claim 31  wherein the surface is a drug delivery system or a diagnostic contrast agent. 
     
     
         33 . The method of  claim 32  wherein the drug delivery system comprises a liposome, a micelle, a nanoparticle, a quantum dot or a dendrimer. 
     
     
         34 . The method of  claim 21  wherein the concentration of the hetero-bifunctional photo-reactive crosslinker in the mixture is about 100 μm to about 400 μm. 
     
     
         35 . The method of  claim 21  wherein the ratio of the immunoglobulin to the hetero-bifunctional photo-reactive crosslinker in the mixture in solution is about 1:10 to about 1:20. 
     
     
         36 . The method of  claim 21  wherein the immunoglobulin is an immunoglobulin fragment comprising the conserved nucleotide binding site. 
     
     
         37 . The method of  claim 36  wherein the immunoglobulin fragment comprising a Fab fragment, a F(ab′) fragment, a F(ab′) 2  fragment, a F v  fragment, a scF v  fragment, a Fd fragment, or a F c  fragment. 
     
     
         38 . An isolated immunoglobulin-ligand conjugate comprising: an immunoglobulin having a conserved nucleotide binding site located away from the antigen binding site of the Fv 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.