Methods for site-specific drug conjugation to antibodies

Abstract

Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve therapeutic index over traditional chemotherapy. Drugs and linkers have been the current focus of ADC development, in addition to antibody and target selection. Recently, however,the importance of conjugate homogeneity has been realized. The current methods for drug attachment lead to a heterogeneous mixture, and some populations of that mixture have poor in vivo performance. New methods for site-specific drug attachment lead to more homogeneous conjugates and allow control of the site of drug attachment. These subtle improvements can have profound effects on in vivo efficacy and therapeutic index. This review examines current methods for site-specific drug conjugation to antibodies, and compares in vivo results with their non-specifically conjugated counterparts. The apparent improvement in pharmacokinetics and the reduced off target toxicity warrant further development of this site-specific modification approach for future ADC development.

Figure 1. Schemes for non-specific (A and B) and site-specific (C, D, and E) drug conjugation to an antibody molecule. (A) After reduction of the interchain disulfide bonds with TCEP or DTT, MC-VC-PAB-MMAE (maleimidocaproyl-valinecitrulline-p-amino-benzyloxycarbonyl-monomethylauristatin E) was attached to the resulting cysteine side chain thiols using the maleimide functional group. The resulting conjugate was a heterogeneous mixture of antibodies with different numbers of drugs attached to the eight cysteine residues involved in interchain disulfide bonds. (B) DM1 (mertansine) was connected to the antibody with a maleimidocyclohexanecarboxylate bifunctional linker (SMCC) through the antibody lysine side chain amines. The resulting conjugate was a heterogeneous mixture with zero to eight drugs per antibody attached to as many as 40 different lysine residues. (C) Site directed mutagenesis was utilized to install one additional cysteine residue in each heavy chain. Upon antibody expression this cysteine was found in a disulfide bond with glutathione. Reduction of all solvent exposed disulfide bonds followed by re-oxidation of the native disulfide bonds with CuSO₄ resulted in a single thiol on each heavy chain which could be modified with a maleimide-containing drug such as MC-VC-PAB-MMAE. This gave a homogeneous product with precisely two drugs attached at the sites of the mutant cysteines. (D) AcLys-VC-PAB-MMAD (acetyllysine-valinecitrulline-p-aminobenzyloxycarbonyl-monomethyldolastatin 10) was attached using the enzyme microbial transglutaminase to catalyze the site-specific reaction between the drug lysine and engineered glutamine side chains in the antibody. This method produced a homogeneous conjugate with two drugs per antibody, attached to the engineered glutamines. (E) The unnatural amino acid p-acetylphenylalanine was genetically incorporated into an antibody using an amber stop codon suppressor tRNA/aaRS pair. The resulting antibody contained one p-acetylphenylalanine in each heavy chain at the location of the genetically encoded amber stop codon. Site-specific modification of p-acetylphenylalanine with AF-oxyamine (auristatin F-oxyamine) resulted in a homogeneous conjugate with precisely two drugs per antibody. (F) Chemical structures and abbreviations of drugs drawn with relevant antibody amino acid side chain attached.