Enzyme‐Mediated Site‐Specific Bioconjugation of Metal Complexes to Proteins: Sortase‐Mediated Coupling of Copper‐64 to a Single‐Chain Antibody

Paterson, Brett M.; Alt, Karen; Jeffery, Charmaine M.; Price, Roger I.; Jagdale, Shweta; Rigby, Sheena; Williams, Charlotte C.; Peter, Karlheinz; Hagemeyer, Christoph E.; Donnelly, Paul S.

doi:10.1002/anie.201402613

Cited by 86 publications

(57 citation statements)

References 41 publications

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“…The feasibility of such a peptide-tagged ADC approach has recently been proven by the introduction of small substrate sequences for enzyme-promoted conjugation at different constant region loops positions of trastuzumab for efficient site-specific ADC preparation16. The possibility to place the peptide-tags at multiple sites within the antibody scaffold would clearly provide an advantage compared to covalent peptide labeling via sortases that exclusively allow for C -terminal payload conjugation17293031. In addition to microbial transglutaminase our approach offers the opportunity for site-specific and efficient antibody conjugation based on a stable isopeptide bond.…”

Section: Resultsmentioning

confidence: 99%

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

Siegmund

Piater

Zakeri

et al. 2016

Sci Rep

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Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.

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Section: Resultsmentioning

confidence: 99%

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

Siegmund

Piater

Zakeri

et al. 2016

Sci Rep

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“…Using such SrtA-mediated reaction, radioactive metal complexes, highly potent anti-tumor drugs, and certain enzymes could be site-specifically labelled to antibodies for different purposes such as in vivo imaging, drug delivery and antibody-antigen detection. [142–145]…”

Section: Antibody Immobilization Chemistriesmentioning

confidence: 99%

Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development

Shen

Rusling

Dixit

2017

Methods

162

109

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Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention. We have discussed the effect of antibody immobilization approaches on the parameters on the performance of an immunoassay.

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“…More recently, Donnelly and coworkers employed SrtA to site-specifically append a GGG-tagged MeCOSar sarcophagine chelator for Cu-64 to an anti-LIBS scFv bearing a C-terminal LPETGG-FLAG tag (Figs. 4 and 6) [28]. In 2013, a particularly creative SrtA-based conjugation strategy was developed to reduce the number of purification steps required to isolate a final conjugate.…”

Section: Peptide Tagsmentioning

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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Enzyme‐Mediated Site‐Specific Bioconjugation of Metal Complexes to Proteins: Sortase‐Mediated Coupling of Copper‐64 to a Single‐Chain Antibody

Cited by 86 publications

References 41 publications

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development

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

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