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June 8, 2017

Mol. Pharmaceutics 2015 Jun 1;12(6):1848-62. doi: 10.1021/acs.molpharmaceut.5b00082. Epub 2015 May 21.

Antibody Conjugates with Unnatural Amino Acids

Trevor J. Hallam, Erik Wold, Alan Wahl, Vaughn V. Smider

Abstract

Antibody conjugates are important in many areas of medicine and biological research, and antibody-drug conjugates (ADCs) are becoming an important next generation class of therapeutics for cancer treatment. Early conjugation technologies relied upon random conjugation to multiple amino acid side chains, resulting in heterogeneous mixtures of labeled antibody. Recent studies, however, strongly support the notion that site-specific conjugation produces a homogeneous population of antibody conjugates with improved pharmacologic properties over randomly coupled molecules. Genetically incorporated unnatural amino acids (uAAs) allow unique orthogonal coupling strategies compared to those used for the 20 naturally occurring amino acids. Thus, uAAs provide a novel paradigm for creation of next generation ADCs. Additionally, uAA-based site-specific conjugation could also empower creation of additional multifunctional conjugates important as biopharmaceuticals, diagnostics, or reagents.

 

PMID:  25898256

DOI: 10.1021/acs.molpharmaceut.5b00082

Full Text: Antibody Conjugates with Unnatural Amino Acids

 

 

Scientific Reports. 2017 April; 7(1):3026. DOI: 10.1038/s41598-017-03192-z. Epub 2017 June 08.

RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates

Gang Yin, Heather T. Stephenson, Junhao Yang, Xiaofan Li, Stephanie M. Armstrong, Tyler H. Heibeck, Cuong Tran, Mary Rose Masikat, Sihong Zhou, Ryan L. Stafford, Alice Y. Yam, John Lee, Alexander R. Steiner, Avinash Gill , Kalyani Penta, Sonia Pollitt, Ramesh Baliga, Christopher J. Murray, Christopher D. Thanos, Leslie M. McEvoy, Aaron K. Sato & Trevor J. Hallam

Abstract

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting
cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

 

PMID: 28596531

PMCID: PMC5465077

DOI: 10.1038/s41598-017-03192-z

Full Text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465077/