Cu-free click cycloaddition reactions in chemical biology

Jewett, John C.; Bertozzi, Carolyn R.

doi:10.1039/b901970g

Cited by 1,496 publications

(1,114 citation statements)

References 46 publications

Supporting

Mentioning

1,104

Contrasting

Unclassified

Order By: Relevance

“…The stability of an anti-Her2 Ab producing stable clone was determined to exceed 60 generations of culture passage based on full-length Ab titers obtained in a 7-d batch culture. In addition to the incorporation of pAF, stable cell lines that incorporate pAZ have also been generated, thereby extending orthogonal site-specific conjugation to 2+3 "click" chemistry (22). We do not yet fully understand how EuCODE stable cell lines can deliver high Ab productivity while at the same time maintaining high cell viability, but we hypothesize that the stably expressed tyrosyl-tRNA/ RS intracellular concentrations were optimized during our cell line development process to balance amber suppression efficiency and cell viability.…”

Section: Resultsmentioning

confidence: 99%

A general approach to site-specific antibody drug conjugates

Tian

Manibusan

et al. 2014

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

286

274

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 99%

A general approach to site-specific antibody drug conjugates

Tian

Manibusan

et al. 2014

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

286

274

View full text Add to dashboard Cite

show abstract

“…Recent findings have established a set of bioorthogonal click reactions that do not require the cytotoxic copper catalyst used in early reports. These copper-free chemistries include strain-promoted azide-alkyne cycloaddition (SPAAC) and the inverse electron demand Diels-Alder reaction between tetrazine and norbornene [24,25]. Previous reports have used these click reactions primarily to crosslink click endfunctionalized branched polyethylene glycol (PEG) with linear crosslinkers composed of either PEG or linear peptides terminated with the appropriate click reaction pair [26][27][28][29].…”

Section: Introductionmentioning

confidence: 99%

Versatile click alginate hydrogels crosslinked via tetrazine–norbornene chemistry

et al. 2015

View full text Add to dashboard Cite

Abstract:Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry.Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.

show abstract

“…[23][24][25][26] The cyclooctyne reagent can be tuned for fast kinetics and the reaction proceeds selectively under a wide range of conditions. [27][28][29][30][31] However, harnessing these qualities for heterobifunctional protein conjugate synthesis first requires a practical route for the site-specific introduction of the necessary reactive partners.…”

mentioning

confidence: 99%