title: Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Phosphopantetheinyl Transferases.
abstract: Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Jan
authors.initials: J
authors.lastname: Grünewald
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Heath E
authors.initials: HE
authors.lastname: Klock
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Cellitti
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Badry
authors.initials: B
authors.lastname: Bursulaya
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: McMullan
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: David H
authors.initials: DH
authors.lastname: Jones
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Hsien-Po
authors.initials: HP
authors.lastname: Chiu
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Xing
authors.initials: X
authors.lastname: Wang
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Paula
authors.initials: P
authors.lastname: Patterson
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Huanfang
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Julie
authors.initials: J
authors.lastname: Vance
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Edward
authors.initials: E
authors.lastname: Nigoghossian
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Hung
authors.initials: H
authors.lastname: Tong
authors.affiliation: Novartis Institutes for BioMedical Research , 4560 Horton Street, Emeryville, California 94608-2916, United States.
authors.forename: Dylan
authors.initials: D
authors.lastname: Daniel
authors.affiliation: Novartis Institutes for BioMedical Research , 4560 Horton Street, Emeryville, California 94608-2916, United States.
authors.forename: William
authors.initials: W
authors.lastname: Mallet
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Weijia
authors.initials: W
authors.lastname: Ou
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Uno
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Ansgar
authors.initials: A
authors.lastname: Brock
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
authors.affiliation: Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.
authors.forename: Bernhard H
authors.initials: BH
authors.lastname: Geierstanger
mesh_terms: D062365:Aminobenzoates / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000378:metabolism*; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*; D000068878:Trastuzumab / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use
chemical_list: D062365:Aminobenzoates; D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D018796:Immunoconjugates; D009842:Oligopeptides; D010455:Peptides; C543533:auristatin; C082662:phosphopantetheinyl transferase; D017855:Transferases (Other Substituted Phosphate Groups); D000068878:Trastuzumab