title: Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.
abstract: Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Shiraishi
authors.forename: Takashige
authors.initials: T
authors.lastname: Muramoto
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Nagatomo
authors.forename: Daisuke
authors.initials: D
authors.lastname: Shinmi
authors.forename: Emiko
authors.initials: E
authors.lastname: Honma
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Masuda
authors.forename: Motoo
authors.initials: M
authors.lastname: Yamasaki
mesh_terms: D045744:Cell Line, Tumor; D003545:Cysteine / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008301:Maleimides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D015202:Protein Engineering; D055550:Protein Stability; D013438:Sulfhydryl Compounds / Q000032:analysis
chemical_list: D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; D008301:Maleimides; D013438:Sulfhydryl Compounds; C043592:maleimide; D011092:Polyethylene Glycols; D003545:Cysteine