title: Synthesis of site-specific antibody-drug conjugates using unnatural amino acids.
abstract: Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2(+) cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Jun Y
authors.initials: JY
authors.lastname: Axup
authors.forename: Krishna M
authors.initials: KM
authors.lastname: Bajjuri
authors.forename: Melissa
authors.initials: M
authors.lastname: Ritland
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Hutchins
authors.forename: Chan Hyuk
authors.initials: CH
authors.lastname: Kim
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Kazane
authors.forename: Rajkumar
authors.initials: R
authors.lastname: Halder
authors.forename: Jane S
authors.initials: JS
authors.lastname: Forsyth
authors.forename: Antonio F
authors.initials: AF
authors.lastname: Santidrian
authors.forename: Karin
authors.initials: K
authors.lastname: Stafin
authors.forename: Yingchun
authors.initials: Y
authors.lastname: Lu
authors.forename: Hon
authors.initials: H
authors.lastname: Tran
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Seller
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Biroc
authors.forename: Aga
authors.initials: A
authors.lastname: Szydlik
authors.forename: Jason K
authors.initials: JK
authors.lastname: Pinkstaff
authors.forename: Feng
authors.initials: F
authors.lastname: Tian
authors.forename: Subhash C
authors.initials: SC
authors.lastname: Sinha
authors.forename: Brunhilde
authors.initials: B
authors.lastname: Felding-Habermann
authors.forename: Vaughn V
authors.initials: VV
authors.lastname: Smider
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D062365:Aminobenzoates / Q000737:chemistry; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D001943:Breast Neoplasms / Q000188:drug therapy*; D045744:Cell Line, Tumor; D055808:Drug Discovery / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005260:Female; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000493:pharmacokinetics / Q000627:therapeutic use; D007074:Immunoglobulin G / Q000737:chemistry; D051379:Mice; D016513:Mice, SCID; D009842:Oligopeptides / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000276:immunology; D000068878:Trastuzumab
chemical_list: D000596:Amino Acids; D062365:Aminobenzoates; D061067:Antibodies, Monoclonal, Humanized; D018796:Immunoconjugates; D007074:Immunoglobulin G; D009842:Oligopeptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C543533:auristatin