Cys80 conjugated immunoglobulins

Abstract

Provided herein are methods for generating conjugated immunoglobulins, the method comprising: decapping a cysteine at amino acid position 80 (“Cys80”) in a light chain variable region of an immunoglobulin, wherein the immunoglobulin comprises a heavy chain variable region and the light chain variable region; and conjugating a thiol-reactive compound to the Cys80, wherein the thiol-reactive compound comprises a thiol-reactive group. Antigen-binding molecules and methods for generating the same, immunoglobulins as well as nucleic acid molecules encoding the immunoglobulins and host cells comprising the nucleic acid molecules, conjugated immunoglobulins, and light chain variable regions for use in a conjugated immunoglobulin are also provided.

What is claimed: 
     
         1 . A method for generating a conjugated immunoglobulin, the method comprising:
 decapping a cysteine at amino acid position 80 (“Cys80”) in a light chain variable region of an immunoglobulin derived from rabbit, wherein the immunoglobulin comprises a heavy chain variable region and the light chain variable region; and   conjugating a thiol-reactive compound to the Cys80, wherein the thiol-reactive compound comprises a thiol-reactive group.   
     
     
         2 . The method of  claim 1 , wherein the decapping comprises incubating the immunoglobulin with a reducing buffer followed by incubating the immunoglobulin with an oxidizing buffer. 
     
     
         3 . The method of  claim 2 , further comprising immobilizing the immunoglobulin on a matrix prior to the incubating with the reducing buffer and eluting the immunoglobulin from the matrix following the incubating with the oxidizing buffer. 
     
     
         4 . The method of  claim 1 , wherein the thiol-reactive compound is attached to a functional agent. 
     
     
         5 . The method of  claim 4 , wherein the functional agent comprises a fluorophore, fluorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug. 
     
     
         6 . The method of  claim 1 , wherein the thiol-reactive compound is bound to a second thiol-reactive compound, the second thiol-reactive compound being bound to a second immunoglobulin having a second heavy chain variable region and a second light chain variable region, the second light chain variable region having a cysteine at amino acid position 80 (“Cys80 2 ”), wherein the second thiol-reactive compound comprises a second thiol-reactive group bound to the Cys80 2 . 
     
     
         7 . The method of  claim 1 , wherein the Cys80 is unpaired. 
     
     
         8 . The method of  claim 1 , further comprising substituting an amino acid at position 83 with an amino acid residue other than Phe, Lys, or Cys. 
     
     
         9 . A method for generating an antigen-binding molecule, the method comprising incubating a first conjugated immunoglobulin with a second conjugated immunoglobulin to generate the antigen-binding molecule, wherein:
 the first conjugated immunoglobulin comprises a first heavy chain variable region and a first light chain variable region, the first light chain variable region having a cysteine at position 80 (“Cys80 1 ”) wherein the Cys80 1  is conjugated to a first thiol-reactive compound comprising a first thiol-reactive group; and   the second conjugated immunoglobulin comprises a second heavy chain variable region and a second light chain variable region, the second light chain variable region having a cysteine at position 80 (“Cys80 2 ”) wherein the Cys80 2  is conjugated to a second thiol-reactive compound comprising a second thiol-reactive group.   
     
     
         10 . The method of  claim 9 , wherein the Cys80 1 , the Cys80 2 , or both, is unpaired. 
     
     
         11 . The method of  claim 9 , further comprising, prior to the incubating step, decapping the Cys80 1 , Cys80 2 , or both; and
 conjugating a first thiol-reactive compound to the Cys80 1 , a second thiol-reactive compound to the Cys80 2 , or both, wherein the first thiol-reactive compound comprises a first thiol-reactive group and the second thiol-reactive compound comprises a second thiol-reactive group.   
     
     
         12 . The method of  claim 9 , wherein the first thiol-reactive compound further comprises a first functional agent, the second thiol-reactive compound further comprises a second functional agent, or both. 
     
     
         13 . The method of  claim 9 , wherein the first immunoglobulin is a first Fab, the second immunoglobulin is a second Fab, or both. 
     
     
         14 . The method of  claim 9 , further comprising substituting an amino acid at position 83 of the first light chain variable region with an amino acid residue other than Phe, Lys, or Cys, substituting an amino acid at position 83 of the second light chain variable region with an amino acid residue other than Phe, Lys, or Cys, or both. 
     
     
         15 . The antigen-binding molecule produced according to the method of  claim 9 . 
     
     
         16 . A antigen-binding molecule comprising:
 a first conjugated immunoglobulin comprising a first heavy chain variable region and a first light chain variable region, the first light chain variable region having a cysteine at position 80 (“Cys80 1 ”), wherein the Cys80 1  is conjugated to a first thiol-reactive compound comprising a first thiol-reactive group, and   a second conjugated immunoglobulin comprising a second heavy chain variable region and a second light chain variable region, the second light chain variable region having a cysteine at position 80 (“Cys80 2 ”) wherein the Cys80 2  is conjugated to a second thiol-reactive compound comprising a second thiol-reactive group.   
     
     
         17 . The antigen-binding molecule of  claim 16 , wherein Cys80 1 , Cys80 2 , or both, is unpaired. 
     
     
         18 . The antigen-binding molecule of  claim 16 , wherein the amino acid at position 83 of the first immunoglobulin, the amino acid at position 83 of the second immunoglobulin, or both, is an amino acid residue other than Phe, Lys, or Cys. 
     
     
         19 . The antigen-binding molecule of  claim 16 , wherein the first thiol-reactive compound further comprises a first functional agent, the second thiol-reactive compound further comprises a second functional agent, or both. 
     
     
         20 . The antigen-binding molecule of  claim 16 , wherein the first immunoglobulin, second immunoglobulin, or both is a Fab.