Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases

Mukai, Takahito; Kobayashi, T.; Hino, Nobumasa; Yanagisawa, Takatoshi; Sakamoto, Kensaku; Yokoyama, Shigeyuki

doi:10.1016/j.bbrc.2008.04.164

Cited by 252 publications

(299 citation statements)

References 30 publications

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“…Die hohe FRET-Effizienz dieser Population ist in Einklang mit der Kristallstruktur von GFP, [22] [23] Andere modifizierte Cyclooctine, wie Dibenzocyclooctine, [24] könnten hingegen wegen ihrer Größe die Synthetase und/oder die Translationsmaschinerie des Wirtes vor beträchtliche Herausforderungen stellen. Da pylRS von M. mazei bekanntermaßen auch orthogonal in einer Vielzahl an eukaryotischen Organismen [19,25] …”

Section: Methodsunclassified

“…Zudem wurde kürzlich gezeigt, dass die Carbamatbindung der Lysinseitenkette ein wichtiger Diskriminator für den erfolgreichen Einbau von UASs ist. [16,19,20] Daher synthetisierten wir, wie in Schema S1 (siehe Hintergrundinformationen) dargestellt, das Lysinderivat 1 in sechs Stufen aus kommerziell erhältlichem Cyclohepten. Der letzte Reaktionsschritt, das Abspalten der tert-Butoxycarbonyl-Schutzgruppe mit 70-proz.…”

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Genetisch kodierte kupferfreie Klick‐Chemie

et al. 2011

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Section: Methodsunclassified

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Genetisch kodierte kupferfreie Klick‐Chemie

et al. 2011

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“…The production of more complex glycosylated proteins, such as full-length antibodies, requires a eukaryotic expression system such as CHO cells. Previous attempts to incorporate nonnative amino acids in eukaryotic organisms have met with limited success as the product titers achieved were not high enough for product development and commercialization (11,12).…”

Section: Significancementioning

confidence: 99%

A general approach to site-specific antibody drug conjugates

Tian

Manibusan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

275

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Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oximebonded, site-specific NDCs were even more apparent when lowantigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.A ntibody drug conjugates (ADCs) are emerging as a new class of anticancer therapeutics that combine the efficacy of small-molecule therapeutics with the targeting ability of an antibody (Ab) (1, 2). By combining these two components into a single molecular entity, highly cytotoxic small-molecule drugs (SMDs) can be delivered to cancerous target tissues, thereby enhancing efficacy while reducing the potential systemic toxic side effects of the SMD. Conventional ADCs are typically produced by conjugating the SMD to the Ab through the side chains of either surface-exposed lysines or free cysteines generated through reduction of interchain disulfide bonds (3, 4). Because antibodies contain many lysine and cysteine residues, conventional conjugation typically produces heterogeneous mixtures that present challenges with respect to analytical characterization and manufacturing. Furthermore, the individual constituents of these mixtures exhibit different pharmacology with respect to their pharmacokinetic, efficacy, and safety profiles, hindering a rational approach to optimizing this modality (5).Recently, it was reported that the pharmacological profile of ADCs may be improved by applying site-specific conjugation technologies that make use of surface-exposed cysteine residues engineered into antibodies (THIOMABS) that are then conjugated to the SMD, resulting in site-specifically conjugated ADCs (TDCs) with defined Ab-drug ratios. Rel...

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“…The ncAA Ne-(tert-butoxycarbonyl)-L-lysine (BocK) (Fig. 1B) is efficiently incorporated by the PylRS in response to the TAG codon (27) in E. coli and was used as a prototypical ncAA at the permissive site K9 of DsRed. We inserted the gene for the DsRed-K9TAG mutant with a His 6 tag at its C terminus into plasmid pRIPP under the control of an IPTG (isopropyl β-D-1-thiogalactopyranoside)-inducible P grac promoter to afford pRIPPDsRed(K9TAG) (Fig.…”

Section: Significancementioning

confidence: 99%

Recombinant thiopeptides containing noncanonical amino acids

Luo

Zambaldo

Liu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus. We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.noncanonical amino acid | biosynthesis | natural products | thiopeptides | antibiotic

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Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases

Cited by 252 publications

References 30 publications

Genetisch kodierte kupferfreie Klick‐Chemie

Genetisch kodierte kupferfreie Klick‐Chemie

A general approach to site-specific antibody drug conjugates

Recombinant thiopeptides containing noncanonical amino acids

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