title: Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Cysteine Insertion.
abstract: Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that combine 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 acids 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 pyrrolobenzodiazepine dimer (SG3249) resulted in comparable efficiency of site-specific conjugation between cysteine-inserted and cysteine-substituted antibodies. Cysteine-inserted ADCs were shown to have biophysical properties, FcRn, and antigen binding affinity similar to the cysteine-substituted ADCs. These ADCs were comparable for serum stability to the ADCs prepared using cysteine-mutagenesis and had selective and potent cytotoxicity against human prostate cancer cells. Two of the cysteine-inserted variants abolish binding of the resulting ADCs to FcγRs in vitro, thereby potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcγRs, which may result in mitigating off-target toxicities. A selected cysteine-inserted ADC demonstrated potent dose-dependent anti-tumor activity in a xenograph tumor mouse model of human breast adenocarcinoma expressing the oncofetal antigen 5T4.
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Nazzareno
authors.identifier: 0000-0001-9379-1646
authors.initials: N
authors.lastname: Dimasi
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Ryan
authors.initials: R
authors.lastname: Fleming
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Haihong
authors.initials: H
authors.lastname: Zhong
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Binyam
authors.initials: B
authors.lastname: Bezabeh
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Krista
authors.initials: K
authors.lastname: Kinneer
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Christie
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Christine
authors.initials: C
authors.lastname: Fazenbaker
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Herren
authors.initials: H
authors.lastname: Wu
authors.affiliation: Antibody Discovery and Protein Engineering and ‡Oncology Research, MedImmune , Gaithersburg, Maryland 20878, United States.
authors.forename: Changshou
authors.initials: C
authors.lastname: Gao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry*; D005260:Female; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000627:therapeutic use; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy; D051379:Mice; D008819:Mice, Nude; D000068878:Trastuzumab / Q000737:chemistry / Q000627:therapeutic use; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D018796:Immunoconjugates; D003545:Cysteine; D000068878:Trastuzumab
keywords: SG3249; antibody engineering; antibody−drug conjugates; cysteine mutagenesis; cysteine-inserted antibody; oncofetal antigen 5T4; pyrrolobenzodiazepine dimers; site-specific conjugation; tesirine