Antibodies and conjugates thereof

Abstract

Provided herein are anti-VEGF-A antibodies and antibody conjugates thereof. Some embodiments of the antibodies can be conjugated to a moiety, such as a HEMA-PC polymer. Some embodiments of the antibody conjugates can retain or enhance antibody activity. The antibody and conjugate thereof can be particularly useful for treating diabetic retinopathy. Further provided are methods for conjugation of a polymer to a protein such as an antibody, such as IgG1.

1 .- 36 . (canceled) 
     
     
         37 . A method of making an antibody conjugate comprising an anti-VEGF-A antibody conjugated to a phosphorylcholine containing polymer, the method comprising the step of:
 conjugating an anti-VEGF-A antibody to a phosphorylcholine containing polymer;   wherein the anti-VEGF-A antibody comprises a cysteine residue added via recombinant DNA technology and wherein the cysteine is outside a variable region of the antibody;
 wherein the phosphorylcholine containing polymer comprises a sulfhydryl specific reacting group selected from the group consisting of a maleimide, a vinylsulfone, an orthopyridyl-disulfide, and an iodoacetamide; and 
   wherein the sulfhydryl specific reacting group on the phosphorylcholine containing polymer reacts with the cysteine residue on the anti-VEGF-A antibody to make the antibody conjugate.   
     
     
         38 . The method according to  claim 37  wherein the anti-VEGF-A antibody is an immunoglobulin G (IgG) and the cysteine is in the Fc region of the antibody. 
     
     
         39 . The method according to  claim 37 , wherein the anti-VEGF-A antibody comprises a light chain and a heavy chain, wherein the anti-VEGF-A antibody heavy chain comprises CDR H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11), and the anti-VEGF-A antibody light chain comprises CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: QQYSTVPWT (SEQ ID NO: 14). 
     
     
         40 . The method according to  claim 38 , wherein the anti-VEGF-A antibody heavy chain isotype is IgG1, wherein the anti-VEGF-A antibody constant domain has one or more mutations relative to an IgG1 constant domain to modulate effector function. 
     
     
         41 .- 43 . (canceled) 
     
     
         44 . The method according to  claim 39 , wherein the anti-VEGF-A antibody heavy chain isotype is IgG1 and comprises the mutations (EU numbering) L234A, L235A, and G237A. 
     
     
         45 . The method according to  claim 44  wherein the cysteine residue added by recombinant DNA technology is selected from the group consisting of Q347C (EU numbering) and L443C (EU numbering). 
     
     
         46 . (canceled) 
     
     
         47 . The method according to  claim 45 , wherein the sequence of the anti-VEGF-A antibody heavy chain is SEQ ID NO: 1 and sequence of the anti-VEGF-A antibody light chain is SEQ ID NO: 2. 
     
     
         48 . The method according to  claim 39 , wherein the sulfhydryl specific reacting group is maleimide. 
     
     
         49 . The method according to  claim 48  wherein the phosphorylcholine containing polymer comprises 2-(methacryloyloxyethyl)-2′-(trimethylammonium)ethyl phosphate (MPC) monomers as set forth below: 
       
         
           
           
               
               
           
         
       
     
     
         50 . The method according to  claim 49 , wherein the polymer has three or more arms. 
     
     
         51 .- 53 . (canceled) 
     
     
         54 . The method according to  claim 50 , wherein the polymer has a molecular weight between about 300,000 and 1,750,000 Da. 
     
     
         55 . (canceled) 
     
     
         56 . (canceled) 
     
     
         57 . The method according to  claim 39 , further comprising the step of contacting the anti-VEGF-A antibody with a thiol reductant under conditions that produce a reduced cysteine sulfhydryl group to produce a reduced anti-VEGF-A antibody in which all cysteine residues are reduced. 
     
     
         58 .- 61 . (canceled) 
     
     
         62 . The method according to  claim 57 , further comprising the steps of removing the thiol reductant from the reduced anti-VEGF-A antibody, and treating the reduced anti-VEGF-A antibody with an oxidizing agent. 
     
     
         63 .- 70 . (canceled) 
     
     
         71 . The method according to  claim 39 , wherein the method further comprises the step of polymerizing a free radically polymerizable phosphorylcholine containing monomer in a polymerization medium to provide the phosphorylcholine containing polymer, the medium comprising:
 the radically polymerizable phosphorylcholine containing monomer;   a transition metal catalyst M t   (q−1)+  wherein M t  is a transition metal, q is the maximum oxidation state of the metal and q−1 is the oxidation state of the metal, wherein the metal can act as a catalyst, wherein the transition metal catalyst is supplied as a salt of the form M t   (q−1)+ X′ (q−1) , wherein X′ is a counterion or group, or wherein the transition metal catalyst is supplied in situ by providing the inactive metal salt at its highest oxidation state M t   q+ X′ q  together with a reducing agent that is capable of reducing the transition metal from the oxidized inactive state to the reduced active state;   a ligand; and   an initiator.   
     
     
         72 . The method according to  claim 71  wherein the radically polymerizable phosphorylcholine containing monomer is 
       
         
           
           
               
               
           
         
         wherein
 R1 is H or C 1-6  alkyl; 
 R2, R3, R4 are each methyl; and 
 X and Y are each 2. 
 
       
     
     
         73 .- 106 . (canceled) 
     
     
         107 . A process for preparing a conjugated protein, the process comprising:
 reducing one or more cysteines in a protein to form a decapped protein in a solution;   reoxidizing the decapped protein to restore at least one disulfide linkage in the reduced protein while ensuring that an engineered cysteine residue in the protein remains in a free thiol form to thereby form a reoxidized decapped protein in the solution;   adding at least one excipient to the solution, wherein the excipient reduces a polymer induced protein precipitation;   adding a polymer to the solution; and   conjugating the polymer to the reoxidized decapped protein at the engineered cysteine residue to form a conjugated protein.   
     
     
         108 . The process of  claim 107 , wherein the protein is an antibody, an antibody protein fusion or a binding fragment thereof. 
     
     
         109 . The process of  claim 108 , wherein the excipient is an acid or a base. 
     
     
         110 . The process of  claim 108 , wherein the excipient is selected from the group consisting of at least one of: a detergent, a sugar, and a charged amino acid. 
     
     
         111 . The process of  claim 107 , wherein reaction of a polymer with the reduced protein occurs under aqueous conditions between pH 6.0 to pH 8.5. 
     
     
         112 .- 121 . (canceled)