Methods for identification of sites for igg conjugation

Abstract

The present disclosure relates to immunoglobulins and immunoglobulin conjugates with reduced oligomerization and efficient labeling and compositions, methods of generating such immunoglobulins and immunoglobulin conjugates and methods of using such immunoglobulin conjugates particularly in the treatment and prevention of disease.

1 . (canceled) 
     
     
         2 : An immunoglobulin conjugate, comprising A) an immunoglobulin having a mutation at a residue selected from the group consisting of 25(V H ), 125(C H1 ), 248(C H2 ), 254(C H2 ), 286(C H2 ), and 326(C H2 ), wherein the residue numbering for the immunoglobulin is according to Kabat numbering, wherein the mutation is a substitution with a cysteine residue, and B) an atom or molecule, wherein the atom or molecule is conjugated to the cysteine residue. 
     
     
         3 : The immunoglobulin conjugate of  claim 2 , further comprising a linker molecule having at least two reactive sites, wherein a first reactive site is bound to the cysteine residue of the immunoglobulin and a second reactive site is bound to the atom or molecule. 
     
     
         4 : The immunoglobulin conjugate of  claim 3 , wherein the linker molecule is selected from the group consisting of a hydrazone, a peptide, a chelating agent, and a maleimide. 
     
     
         5 : The immunoglobulin conjugate of  claim 3 , wherein the linker molecule forms a disulfide linkage with the cysteine residue. 
     
     
         6 : The immunoglobulin conjugate of  claim 2 , wherein the atom or molecule is selected from the group consisting of a radionuclide, a chemotherapeutic agent, a microbial toxin, a plant toxin, a polymer, a carbohydrate, a cytokine, a fluorescent label, a luminescent label, an enzyme-substrate label, an enzyme, a peptide, a peptidomimetic, a nucleotide, an siRNA, a microRNA, an RNA mimetic, and an aptamer. 
     
     
         7 : The immunoglobulin conjugate of  claim 2 , wherein the atom or molecule is selected from the group consisting of  90 Y,  131 I,  67 Cu,  177 Lu,  213 Bi,  211 At, a calicheamicin, a duocarmycin, a maytanisoid, an auristatin, an anthracyclin,  Pseudomonas  exotoxin A, Diphtheria toxin, ricin, polyethylene glycol, hydroxyethyl starch, and a mannosyl residue. 
     
     
         8 : A modified or isolated immunoglobulin comprising a mutation at a residue selected from the group consisting of 25(V H ), 125(C H1 ), 248(C H2 ), 254(C H2 ), 286(C H2 ), and 326(C H2 ), wherein the residue numbering for the immunoglobulin is according to Kabat numbering, wherein the mutation is a substitution with a cysteine residue. 
     
     
         9 : An isolated or recombinant polynucleotide encoding the immunoglobulin of  claim 8 . 
     
     
         10 : A vector comprising the polynucleotide of  claim 9  operably linked to an inducible promoter. 
     
     
         11 : A host cell comprising the vector of  claim 10 . 
     
     
         12 : A method of producing an immunoglobulin, comprising:
 (a) providing a culture medium comprising the host cell of  claim 11 ; and   (b) placing the culture medium in conditions under which the immunoglobulin is expressed.   
     
     
         13 : A method of producing an immunoglobulin conjugate, comprising:
 (a) providing the immunoglobulin of  claim 8 ;   (b) reducing the one or more substituted cysteine residues with a reducing agent to form reduced cysteine residues; and   (c) incubating the immunoglobulin with an atom or molecule, wherein the atom or molecule is reactive with the reduced cysteine residues, to form an immunoglobulin conjugate.   
     
     
         14 : A method for reducing the cross-linking between surface-exposed cysteines of an immunoglobulin in a highly concentrated pharmaceutical formulation of immunoglobulin conjugates, comprising:
 (a) providing an immunoglobulin;   (b) substituting a residue selected from the group consisting of 25(V H ), 125(C H1 ), 248(C H2 ), 254(C H2 ), 286(C H2 ), and 326(C H2 ) with a cysteine residue, wherein the residue numbering for the immunoglobulin is according to Kabat numbering,   (c) reducing the one or more substituted cysteine residues with a reducing agent to form reduced cysteine residues;   (d) incubating the immunoglobulin with an atom or molecule, wherein the molecule is reactive with the reduced cysteine residues, to form an immunoglobulin conjugate; and   (e) generating a highly concentrated, liquid formulation of the immunoglobulin conjugate wherein the immunoglobulin conjugate concentration is at least 20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml.   
     
     
         15 : The method of  claim 14 , wherein the immunoglobulin conjugate comprises an antigen binding activity and the activity is at least eighty percent, at least ninety percent, at least one hundred percent, at least one hundred ten percent, at least one hundred twenty percent, or at least one hundred thirty percent of the antigen binding activity of the unmutated immunoglobulin. 
     
     
         16 : A pharmaceutical composition comprising the immunoglobulin conjugate of  claim 2  and a pharmaceutically acceptable excipient, wherein at least eighty percent, at least eighty-five percent, at least ninety percent, at least ninety-five percent, at least ninety-six percent, at least ninety-seven percent, at least ninety-eight percent, or at least ninety-nine percent of the immunoglobulin conjugate is non-oligomerized monomer. 
     
     
         17 : The pharmaceutical composition of  claim 16 , wherein the immunoglobulin conjugate is at a concentration of at least 10 mg/ml, at least 20 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml, at least 125 mg/ml, or at least 150 mg/ml.