Efficient Preparation of Site-Specific Antibody–Drug Conjugates Using Phosphopantetheinyl Transferases
| Autor: | Weijia Ou, Susan E. Cellitti, Bernhard H. Geierstanger, Paula Patterson, Xing Wang, Heath E. Klock, Edward Nigoghossian, Ansgar Brock, Hung Tong, Dylan Daniel, Jan Grünewald, Badry Bursulaya, William Mallet, David H. Jones, Tetsuo Uno, Daniel McMullan, Hsien-Po Chiu, Julie Vance, Huanfang Zhou, Scott A. Lesley |
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| Rok vydání: | 2015 |
| Předmět: |
Immunoconjugates
Biomedical Engineering Mice Nude Transferases (Other Substituted Phosphate Groups) Pharmaceutical Science Antineoplastic Agents Bioengineering medicine.disease_cause Substrate Specificity Serine Mice Residue (chemistry) Bacterial Proteins Pharmacokinetics Neoplasms medicine Animals Humans Aminobenzoates Pharmacology Oligopeptide biology Chemistry Toxin Organic Chemistry Trastuzumab body regions Biochemistry Cell culture biology.protein Antibody Peptides Oligopeptides Biotechnology Conjugate |
| Zdroj: | Bioconjugate Chemistry. 26:2554-2562 |
| ISSN: | 1520-4812 1043-1802 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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