Site‐Specific Conjugation of ScFvs Antibodies to Nanoparticles by Bioorthogonal Strain‐Promoted Alkyne–Nitrone Cycloaddition

Colombo, Miriam; Sommaruga, Silvia; Mazzucchelli, Serena; Polito, Laura; Verderio, Paolo; Galeffi, Patrizia; Corsi, Fabio; Tortora, Paolo; Prosperi, Davide

doi:10.1002/anie.201106775

Cited by 68 publications

(42 citation statements)

References 39 publications

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“…18−20 Similarly, antibody fragments such as scFV and antibody alternatives such as affibodies have also been cloned and expressed in part to simplify site-specific attachment. 21,22 However, these agents often suffer from decreased stability, short serum half-life, and weak antigen binding. Alternative conjugation strategies for full-length antibodies, such as those that attach conjugates onto glycosylated residues, avoid the need for cloning, and can accelerate the time needed to produce a functional conjugate.…”

Section: Introductionmentioning

confidence: 99%

Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG

Hui

Tsourkas

2014

Bioconjugate Chem.

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Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody’s antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community.

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Section: Introductionmentioning

confidence: 99%

Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG

Hui

Tsourkas

2014

Bioconjugate Chem.

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“…The first generation is represented by nanomaterials functionalized through basic surface chemistries to assess biocompatibility, enhance cellular uptake, and reduce toxicity. The second generation is focused on nanomaterials with optimized surface boundaries that improve stability and targeting in biological systems . These studies were characterized by two important tasks: “stealthiness” and active targeting.…”

Section: Introductionmentioning

confidence: 99%

Delivering Colloidal Nanoparticles to Mammalian Cells: A Nano–Bio Interface Perspective

Verderio

Avvakumova

Alessio

et al. 2014

Adv Healthcare Materials

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Understanding the behavior of multifunctional colloidal nanoparticles capable of biomolecular targeting remains a fascinating challenge in materials science with dramatic implications in view of a possible clinical translation. In several circumstances, assumptions on structure-activity relationships have failed in determining the expected responses of these complex systems in a biological environment. The present Review depicts the most recent advances about colloidal nanoparticles designed for use as tools for cellular nanobiotechnology, in particular, for the preferential transport through different target compartments, including cell membrane, cytoplasm, mitochondria, and nucleus. Besides the conventional entry mechanisms based on crossing the cellular membrane, an insight into modern physical approaches to quantitatively deliver nanomaterials inside cells, such as microinjection and electro-poration, is provided. Recent hypotheses on how the nanoparticle structure and functionalization may affect the interactions at the nano-bio interface, which in turn mediate the nanoparticle internalization routes, are highlighted. In addition, some hurdles when this small interface faces the physiological environment and how this phenomenon can turn into different unexpected responses, are discussed. Finally, possible future developments oriented to synergistically tailor biological and chemical properties of nanoconjugates to improve the control over nanoparticle transport, which could open new scenarios in the field of nanomedicine, are addressed.

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“…Magnetic nanoparticles ( MNP ) were synthesized in organic solvent by solvothermal decomposition as described in our previous work [30]. The 8 nm iron oxide nanoparticles coated with oleic acid dissolved in chloroform (20 mg, 6.7 mL) were treated with an aqueous solution of tetramethylammonium hydroxide (TMAOH, 0.6 g in 20 ml) overnight under vigorous stirring at room temperature [27,30,39].…”

Section: Methodsmentioning

confidence: 99%

“…The 8 nm iron oxide nanoparticles coated with oleic acid dissolved in chloroform (20 mg, 6.7 mL) were treated with an aqueous solution of tetramethylammonium hydroxide (TMAOH, 0.6 g in 20 ml) overnight under vigorous stirring at room temperature [27,30,39]. The organic solution containing the surfactant was removed by centrifugation at 1000 rpm (3 min).…”

Section: Methodsmentioning

confidence: 99%

Immobilization of carboxypeptidase from Sulfolobus solfataricuson magnetic nanoparticles improves enzyme stability and functionality in organic media

et al. 2014

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BackgroundSuperparamagnetic iron oxide nanoparticles (MNP) offer several advantages for applications in biomedical and biotechnological research. In particular, MNP-based immobilization of enzymes allows high surface-to-volume ratio, good dispersibility, easy separation of enzymes from the reaction mixture, and reuse by applying an external magnetic field. In a biotechnological perspective, extremophilic enzymes hold great promise as they often can be used under non-conventional harsh conditions, which may result in substrate transformations that are not achievable with normal enzymes. This prompted us to investigate the effect of MNP bioconjugation on the catalytic properties of a thermostable carboxypeptidase from the hyperthermophilic archaeon Sulfolobus solfataricus (CPSso), which exhibits catalytic properties that are useful in synthetic processes.ResultsCPSso was immobilized onto silica-coated iron oxide nanoparticles via NiNTA-His tag site-directed conjugation. Following the immobilization, CPSso acquired distinctly higher long-term stability at room temperature compared to the free native enzyme, which, in contrast, underwent extensive inactivation after 72 h incubation, thus suggesting a potential utilization of this enzyme under low energy consumption. Moreover, CPSso conjugation also resulted in a significantly higher stability in organic solvents at 40°C, which made it possible to synthesize N-blocked amino acids in remarkably higher yields compared to those of free enzyme.ConclusionsThe nanobioconjugate of CPSso immobilized on silica-coated magnetic nanoparticles exhibited enhanced stability in aqueous media at room temperature as well as in different organic solvents. The improved stability in ethanol paves the way to possible applications of immobilized CPSso, in particular as a biocatalyst for the synthesis of N-blocked amino acids. Another potential application might be amino acid racemate resolution, a critical and expensive step in chemical synthesis.

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Site‐Specific Conjugation of ScFvs Antibodies to Nanoparticles by Bioorthogonal Strain‐Promoted Alkyne–Nitrone Cycloaddition

Cited by 68 publications

References 39 publications

Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG

Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG

Delivering Colloidal Nanoparticles to Mammalian Cells: A Nano–Bio Interface Perspective

Immobilization of carboxypeptidase from Sulfolobus solfataricuson magnetic nanoparticles improves enzyme stability and functionality in organic media

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