title: Application of strain-promoted azide-alkyne cycloaddition and tetrazine ligation to targeted Fc-drug conjugates.
abstract: We have previously described an approach whereby antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec) are directed against a variety of targets by changing the peptide or small molecule to which they are conjugated. In the present work, we describe methodology for improving the efficacy of these Fc-Sec conjugates by incorporating cytotoxic drugs. The Fc-Sec protein is first programmed to target specific tumor cell types by attachment of a bifunctional linker that contains a "clickable" handle (e.g., cyclobutane or cyclooctyne) in addition to a tumor cell-binding peptide or small molecule. Following Fc-Sec conjugation, a cytotoxic warhead is then attached by cycloaddition reactions of tetrazine or azide-containing linker. To validate this approach, we used a model system in which folic acid (FA) is the targeting moiety and a disulfide-linked biotin moiety serves as a cytotoxic drug surrogate. We demonstrated successful targeting of Fc-Sec proteins to folate-receptor expressing tumor cells. Tetrazine ligation was found to be an efficient method for biotin "arming" of the folate-targeted Fc-Sec proteins. We also report novel bioconjugation methodologies that use [4 + 2] cycloaddition reactions between tetrazines and cyclooctynes.
authors.affiliation: Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Thomas
authors.forename: Huiting
authors.initials: H
authors.lastname: Cui
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: North
authors.forename: Thomas
authors.initials: T
authors.lastname: Hofer
authors.forename: Christoph
authors.initials: C
authors.lastname: Rader
authors.forename: Terrence R
authors.initials: TR
authors.lastname: Burke
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology; D001386:Azides / Q000737:chemistry*; D061565:Cycloaddition Reaction; D003503:Cyclobutanes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D017279:Selenocysteine / Q000737:chemistry; D013777:Tetrazoles / Q000737:chemistry*
chemical_list: D000480:Alkynes; D000970:Antineoplastic Agents; D001386:Azides; D003503:Cyclobutanes; D007141:Immunoglobulin Fc Fragments; D013777:Tetrazoles; D017279:Selenocysteine