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: Jan Grünewald, Heath E Klock, Susan E Cellitti, Badry Bursulaya, Daniel McMullan, David H Jones, Hsien-Po Chiu, Xing Wang, Paula Patterson, Huanfang Zhou, Julie Vance, Edward Nigoghossian, Hung Tong, Dylan Daniel, William Mallet, Weijia Ou, Tetsuo Uno, Ansgar Brock, Scott A Lesley, Bernhard H Geierstanger
• Journal: Bioconjugate chemistry (Bioconjug Chem) Vol. 26 Issue 12 Pg. 2554-62 (Dec 16 2015) ISSN: 1520-4812 [Electronic] United States
• PMID: 26588668 (Publication Type: Journal Article)

Chemical References

• Aminobenzoates
• Antineoplastic Agents
• Bacterial Proteins
• Immunoconjugates
• Oligopeptides
• Peptides
• auristatin
• phosphopantetheinyl transferase
• Transferases (Other Substituted Phosphate Groups)
• Trastuzumab

Topics

• Aminobenzoates (chemistry, metabolism, therapeutic use)
• Animals
• Antineoplastic Agents (chemistry, metabolism, therapeutic use)
• Bacterial Proteins (metabolism)
• Humans
• Immunoconjugates (chemistry, metabolism, therapeutic use)
• Mice
• Mice, Nude
• Neoplasms (drug therapy, metabolism)
• Oligopeptides (chemistry, metabolism, therapeutic use)
• Peptides (chemistry, metabolism)
• Substrate Specificity
• Transferases (Other Substituted Phosphate Groups) (metabolism)
• Trastuzumab (chemistry, metabolism, therapeutic use)