Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Cysteine Insertion

Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that comhine the specificity of antibodies with the high-potency Of cytotoxic drugs., Engineering cysteine residues in the antibodies using mutagenesis is a common method to prepare site-specific ADCs. With this approach, solvent accessible amino acid's in the antibody have been:selected for substitution with cysteine for conjugating maleimide-bearing cytotoxic drugs) resulting in homogeneous and stable site-specific ADCS. Here we describe a cysteine engineering approach based on the insertion of,cysteines,before and after selected sites in the antibody, which can be used for site-specific preparation of ADCs. cysteine-inserted antibodies have expression level and monomeric content similar to the native antibodies. Conjugation to a pyrrolobenizodiazepine dinner (SG3249) resulted in, comparable efficiency of site-specific conjugation between cysteine-inserted and tysteine-subStituted.antibodies. Cysteine-itisertecrADCs were shown to have biophysical propertieS, FcPn, and antigen binding affinity similar to the eysteine-substituted ADCs. These ADCs were comparable for serum stability to the ADCs prepared using cysteitie-mutagenesis and had selective and potent cytotoxicity against human prostate Cancer cells. Two of the cysteine-iuserted variants abolish binding of the resulting ADCs to FcyRs in vitro, thereby potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcyRs, which may result in mitigating off -target toxicities. A selected cysteine-inserted.ADC demonstrated potent dose-dependent antitumor activity in a xenograph tumor mouse model of human breast adenocarcinorna expressing the oncofetal antigen 5T4.