RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates
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 i...
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2017
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oai:doaj.org-article:847eaf6ce18d47c0aa91ced7f6902fb02021-12-02T12:32:14ZRF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates10.1038/s41598-017-03192-z2045-2322https://doaj.org/article/847eaf6ce18d47c0aa91ced7f6902fb02017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03192-zhttps://doaj.org/toc/2045-2322Abstract 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.Gang YinHeather T. StephensonJunhao YangXiaofan LiStephanie M. ArmstrongTyler H. HeibeckCuong TranMary Rose MasikatSihong ZhouRyan L. StaffordAlice Y. YamJohn LeeAlexander R. SteinerAvinash GillKalyani PentaSonia PollittRamesh BaligaChristopher J. MurrayChristopher D. ThanosLeslie M. McEvoyAaron K. SatoTrevor J. HallamNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017) |
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DOAJ |
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Medicine R Science Q |
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Medicine R Science Q 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 RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates |
| description |
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. |
| format |
article |
| author |
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 |
| author_facet |
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 |
| author_sort |
Gang Yin |
| title |
RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates |
| title_short |
RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates |
| title_full |
RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates |
| title_fullStr |
RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates |
| title_full_unstemmed |
RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates |
| title_sort |
rf1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates |
| publisher |
Nature Portfolio |
| publishDate |
2017 |
| url |
https://doaj.org/article/847eaf6ce18d47c0aa91ced7f6902fb0 |
| work_keys_str_mv |
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