Antibody–Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents

Dovgan, Igor; Koniev, Oleksandr; Kolodych, Sergii; Wagner, Alain

doi:10.1021/acs.bioconjchem.9b00306

Cited by 105 publications

(101 citation statements)

References 178 publications

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“…Antibodies are considered one of the most effective biomolecules to be used for detection methodologies and applications [42][43][44]. Due to their particular characteristics, such as high affinity and specificity, a large variety of immunoassay formats implementing antibodies as reagents have been developed for disease diagnoses.…”

Section: Discussionmentioning

confidence: 99%

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

Zhao

et al. 2020

J Nanobiotechnol

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Background: Antibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV.Results: In the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni-NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests. Conclusions:For the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article' s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article'

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Section: Discussionmentioning

confidence: 99%

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

Zhao

et al. 2020

J Nanobiotechnol

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“…However, similar molecular calculus may be applied to extensively cleared molecular entities which may otherwise have trouble reaching the tissues of interest. A rapidly-advancing molecular space which is typically impeded by delivery issues is that of synthetic therapeutic oligonucleotides including antisense oligonucleotides (ASOs) and short interfering RNA (siRNA) [107][108][109].…”

Section: Nucleic Acid Conjugatesmentioning

confidence: 99%

“…While this Fab'-based study utilized structural cystines for conjugation, other therapeutic antibody-oligonucleotide conjugation systems not yet described in this section have been reported, including two oncology examples which applied two-step conjugations for azide-labeling the antibody which were then treated with cyclooctyne-appended ASOs to generate the conjugates [121,122]. Other antibody-oligonucleotide conjugation methods beyond the scope of this review (i.e., for immuno PCR applications, pre-targeted radiotherapy, or those which utilize non-covalent heterogeneous complexes) have been recently reviewed [109].…”

Section: Nucleic Acid Conjugatesmentioning

confidence: 99%

Antibody Conjugates-Recent Advances and Future Innovations

et al. 2020

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Monoclonal antibodies have evolved from research tools to powerful therapeutics in the past 30 years. Clinical success rates of antibodies have exceeded expectations, resulting in heavy investment in biologics discovery and development in addition to traditional small molecules across the industry. However, protein therapeutics cannot drug targets intracellularly and are limited to soluble and cell-surface antigens. Tremendous strides have been made in antibody discovery, protein engineering, formulation, and delivery devices. These advances continue to push the boundaries of biologics to enable antibody conjugates to take advantage of the target specificity and long half-life from an antibody, while delivering highly potent small molecule drugs. While the “magic bullet” concept produced the first wave of antibody conjugates, these entities were met with limited clinical success. This review summarizes the advances and challenges in the field to date with emphasis on antibody conjugation, linker-payload chemistry, novel payload classes, absorption, distribution, metabolism, and excretion (ADME), and product developability. We discuss lessons learned in the development of oncology antibody conjugates and look towards future innovations enabling other therapeutic indications.

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“…Another major area of application is different detection approaches encompassing mainly immuno-polymerase chain reaction (PCR), proximity ligation/extension assays, electrochemical proximity assay, and DNA-PAINT imaging first and foremost by their exceptional sensitivity. The higher number of potential applications of antibody-oligonucleotide conjugates and antibody-oligonucleotide conjugation techniques have been recently reviewed by Dovgan et al 2 …”

Section: Introductionmentioning

confidence: 99%

Site-Specific Linking of an Oligonucleotide to Mono- and Bivalent Recombinant Antibodies with SpyCatcher-SpyTag System for Immuno-PCR

Kushnarova-Vakal¹,

Äärelä²,

Huovinen³

et al. 2020

ACS Omega

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Antibody-oligonucleotide conjugates (AOCs) are a versatile class of chimeric biomolecules for therapeutics and biotechnological applications. Most widely employed chemical labeling methods for proteins are based on targeting of Lys or Cys residues that leads to mixed stoichiometry in the degree of conjugation and may interfere with antigen binding, thus, compromising the function of the antibody. A site-specific oligonucleotide conjugation technology providing full control over valency in mild reaction conditions would be an advancement to the state-of-the-art in bioconjugation. Herein, we demonstrate the production of single-chain variable fragment antibodies with fused SpyCatcher (scFv-SpyCatcher, monovalent) and alkaline phosphatase-SpyCatcher (scFv-AP-SpyCatcher, bivalent) on C-terminus and their conjugation to SpyTag002-oligonucleotide in phosphate-buffered saline (PBS). The formation of a covalent isopeptide bond between the protein and SpyTag002-oligonucleotide was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the functionality of the obtained AOCs was confirmed in immuno-polymerase chain reaction (PCR) assays for the detection of microcystin-LR and 17β-estradiol. Based on time-resolved fluorescence immunoassays with scFv-AP fusion constructs, we observed that the SpyCatcher and SpyCatcher-SpyTag002-oligonucleotide part lowered the absolute signal obtained from the assay by 27.6 and 48.4% at 2 nM and by 26.2 and 27.6% at 100 pM microcystin-LR and 17β-estradiol concentrations, respectively. Nevertheless, the overall sensitivity of the immuno-PCR assays was similar to the time-resolved fluorescence immunoassays performed with the same components. In this study, vectors for SpyCatcher-fusion construction were created for directional cloning with Sfi I sites enabling the rapid generation of AOC constructs for site-specific SpyTag-oligonucleotide conjugation.

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Antibody–Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents

Cited by 105 publications

References 178 publications

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

Antibody Conjugates-Recent Advances and Future Innovations

Site-Specific Linking of an Oligonucleotide to Mono- and Bivalent Recombinant Antibodies with SpyCatcher-SpyTag System for Immuno-PCR

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