Site-Specific Dual Antibody Conjugation via Engineered Cysteine and Selenocysteine Residues

Li, Xiuling; Patterson, James T.; Sarkar, Mohosin; Pedzisa, Lee; Kodadek, Thomas; Roush, William; Rader, Christoph

doi:10.1021/acs.bioconjchem.5b00244

Cited by 50 publications

(38 citation statements)

References 25 publications

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“…Maleimide and iodoacetamide derivatives of fluorescein were conjugated to humanized anti-human HER2 mAb trastuzumab in scFv-Fc format with a C -terminal Sec residue (Li et al, 2015) using optimized conjugation conditions reported previously (Li et al, 2014) and the resulting selenomab-fluorescein conjugates were incubated in human plasma at 37° C. The maleimide-selenol conjugate at this site was unstable in human plasma and revealed a rapid exchange reaction with human serum albumin (HSA) (Figure 1). This exchange reaction between a succinimide selenoether and a succinimide thioether likely proceeded through a retro-Michael/Michael reaction sequence (Alley et al, 2008; Shen et al, 2012).…”

Section: Resultsmentioning

confidence: 98%

“…Our previous studies exclusively used first-generation selenomabs with one (Cui et al, 2012; Hofer et al, 2009; Hofer et al, 2008; Li et al, 2015; Patterson et al, 2014; Thomas et al, 2012; Thomas et al, 2008; Vire et al, 2014) or two C -terminal Sec residues (Li et al, 2014). This position mirrors the C -terminal location of the Sec residue in the natural selenoprotein thioredoxin reductase 1 (TXNRD1).…”

Section: Discussionmentioning

confidence: 99%

“…Using drug surrogates, we subsequently provided evidence that selenomabs may be suitable for the generation of homogeneous ADCs (Hofer et al, 2009; Li et al, 2015; Li et al, 2014; Patterson et al, 2014; Thomas et al, 2012). In eukaryotes, Sec (also known as the 21st natural amino acid) (Hatfield and Gladyshev, 2002) is encoded by the stop codon UGA and its translational incorporation requires the presence of a Sec incorporation sequence (SECIS) in the 3′ untranslated region (UTR) of the mRNA.…”

Section: Introductionmentioning

confidence: 99%

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Stable and Potent Selenomab-Drug Conjugates

Nelson

Nair

et al. 2017

Cell Chemical Biology

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Summary Selenomabs are engineered monoclonal antibodies with one or more translationally incorporated selenocysteine residues. The unique reactivity of the selenol group of selenocysteine permits site-specific conjugation of drugs. Compared to other natural and unnatural amino acid and carbohydrate residues that have been used for the generation of site-specific antibody-drug conjugates, selenocysteine is particularly reactive, permitting fast, single-step, and efficient reactions under near physiological conditions. Using a tailored conjugation chemistry, we generated highly stable selenomab-drug conjugates and demonstrated their potency and selectivity in vitro and in vivo. These site-specific antibody-drug conjugates built on a selenocysteine interface revealed broad therapeutic utility in liquid and solid malignancy models.

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Section: Resultsmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Stable and Potent Selenomab-Drug Conjugates

Nelson

Nair

et al. 2017

Cell Chemical Biology

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“…While a fluorescein-oxadiazole sulfone probe (FL-ODA ) 18 , based on a phenyl oxadiazole coupling partner (Scheme 4) has been shown to selectively conjugate selenocysteine in antibody applications, the selectivity for allene conjugation with Sec is as of yet unknown. 26, 29 However, the allene electrophiles have been shown to conjugate cysteine with high efficiency. 32 In order to determine the selectivity of allene conjugation and establish whether allenes might be useful for SELENOMAB conjugation, we synthesized an allene-based fluorescein reactive probe 19 (Scheme 4, Fl-ALL).…”

Section: Resultsmentioning

confidence: 99%

Assessment of reagents for selenocysteine conjugation and the stability of selenocysteine adducts

Pedzisa

Rader

et al. 2016

Org. Biomol. Chem.

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Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures that have poor pharmacokinetic properties and decreased efficacy relative to homogenous ADCs. Furthermore, ADCs that are maleimide-based often have inadequate circulatory stability, which can result in premature drug release with consequent off-target toxicities. Selenocysteine-modified antibodies have been developed that allow site-specific antibody conjugation, yielding homogeneous ADCs. Herein, we survey several electrophilic functional groups that react with selenocystine with high efficiency. Several of these result in conjugates with stabilites that are superior to maleimide conjugates. Among these, the allenamide functional group reacts with notably high efficiency, leads to conjugates with remarkable stability, and shows exquisite selectivity for selenocysteine conjugation.

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“…Along these lines, Li et al . have recently produced a scFv-Fc fragment of trastuzumab bearing two engineered cysteines and one selenocysteine in the Fc region [63]. After reduction, the selenocysteine residue of the fragment was selectively coupled to a methyl sulfone-bearing variant of biotin at pH 5.2, while the cysteine residues were conjugated to a methyl sulfone-containing fluorescein moiety at pH 7.4.…”

Section: Unnatural Amino Acidsmentioning

confidence: 99%

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

et al. 2016

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Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification strategies covered in both parts of the review and offer a brief discussion of the overall direction of the field.

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Site-Specific Dual Antibody Conjugation via Engineered Cysteine and Selenocysteine Residues

Cited by 50 publications

References 25 publications

Stable and Potent Selenomab-Drug Conjugates

Stable and Potent Selenomab-Drug Conjugates

Assessment of reagents for selenocysteine conjugation and the stability of selenocysteine adducts

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

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