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Antibodies 2017, 6(4), 20;

Pyrrolobenzodiazepine Antibody-Drug Conjugates Designed for Stable Thiol Conjugation

Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, MD 20878, USA
Spirogen Inc., London, E1 2AX, UK
Oncology Research, MedImmune, Gaithersburg, MD 20878, USA
Authors to whom correspondence should be addressed.
Received: 17 October 2017 / Revised: 20 November 2017 / Accepted: 21 November 2017 / Published: 28 November 2017
(This article belongs to the Special Issue Antibody Drug Conjugates)
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Thiosuccinimide-linked antibody-drug conjugates (ADCs) are susceptible to drug loss over time due to a retro-Michael reaction, which can be prevented by selecting stable conjugation positions or hydrolysis of the thiosuccinimide. Here, we investigate pyrrolobenzodiazepine (PBD) ADC drug-linkers equipped with N-phenyl maleimide functionality for stable thiol conjugation via thiosuccinimide hydrolysis. Two PBD drug-linker formats (enzyme-cleavable and non-cleavable) were evaluated following site-specific conjugation to an engineered cysteine incorporated at position T289, which is known to be unstable for N-alkyl maleimide conjugates. N-phenyl maleimide PBDs conjugated to antibodies with similar efficiencies as N-alkyl maleimide PBDs and enhanced thiosuccinimide hydrolysis for N-phenyl maleimide PBDs was confirmed by mass spectrometry, capillary isoelectric focusing, and a SYPRO Orange dye binding assay. All of the PBD ADCs were highly potent in vitro regardless of maleimide- or linker-type, exhibiting low pM EC50 values. Thiol conjugation to N-phenyl maleimide PBD minimized the retro-Michael reaction in both rat and mouse serum. However, cleavage of the valine-alanine dipeptide in mouse serum for ADCs containing cleavable drug-linker led to drug loss regardless of maleimide type, which impacted ADC potency in tumor growth inhibition studies that were conducted in mouse models. Therapeutic improvement in mouse tumor models was realized for ADCs prepared with non-cleavable PBD drug-linkers that were conjugated through N-phenyl maleimide, where a stronger tumor growth inhibition (TGI) response was achieved when compared to the analogous N-alkyl maleimide drug-linker ADC. Altogether, our findings highlight the stability and efficacy benefits of N-phenyl maleimide functionality for ADCs that are produced with thiol-maleimide conjugation chemistry. View Full-Text
Keywords: PBD dimer; thiosuccinimide hydrolysis; N-phenyl maleimide; retro-Michael reaction; serum stability; ADC; site-specific conjugation PBD dimer; thiosuccinimide hydrolysis; N-phenyl maleimide; retro-Michael reaction; serum stability; ADC; site-specific conjugation

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Christie, R.J.; Tiberghien, A.C.; Du, Q.; Bezabeh, B.; Fleming, R.; Shannon, A.; Mao, S.; Breen, S.; Zhang, J.; Zhong, H.; Harper, J.; Wu, H.; Howard, P.W.; Gao, C. Pyrrolobenzodiazepine Antibody-Drug Conjugates Designed for Stable Thiol Conjugation. Antibodies 2017, 6, 20.

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