In vivo biotinylation and incorporation of a photo‐inducible unnatural amino acid to an antibody‐binding domain improve site‐specific labeling of antibodies

Kanje, Sara; Hober, Sophia

doi:10.1002/biot.201400808

Cited by 20 publications

(30 citation statements)

References 29 publications

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“…For example, bound Z dissociates within 5 min, meaning that any intended antibody modification would be lost on the same time scale . The bound complex may be crosslinked by incorporating an unnatural amino acid with a photoactivatable side chain in ABD and exposing the complex to UV light . It is challenging to produce a uniformly crosslinked complex because the efficiency of photocrosslinking is low and the complex can dissociate during UV treatment, although incomplete conjugation does not interfere with some applications, such as immunohistochemistry .…”

Section: Introductionmentioning

confidence: 99%

High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody‐Binding Domains

Zhou

Kroetsch

Nguyen

et al. 2019

Biotechnology Journal

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Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody-binding protein as an adapter can simplify antibody functionalization by forming a specific antibody-bound complex and introducing site-specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody-binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein ("lasso"), which binds human immunoglobulin G1 (IgG1) with K D = 0.53 nM and a dissociation rate that is 55to 84-fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme-linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12-fold. The small size of the lasso and a long half-life of dissociation make the peptide a useful tool in antibody detection and immobilization.

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Section: Introductionmentioning

confidence: 99%

High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody‐Binding Domains

Zhou

Kroetsch

Nguyen

et al. 2019

Biotechnology Journal

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“…Human antibodies biotinylated with this domain showed a higher binding response when immobilized on a streptavidin surface in an SPR setup, compared to the same antibody that was randomly labeled using NHS-chemistry, and could also successfully be used for detection in an ELISA experiment. 28 From the same research group, a C2 domain with abolished Fc binding, through the N35W and D40T mutations, was used for labeling of antibodies site-specifically at the Fab fragment. Introducing BPA at position 18 enabled 48–64% cross-linking to mouse IgG heavy chains depending on subclass, while BPA at position 29 provided 43–58% cross-linking to human IgG heavy chains depending on subclass.…”

Section: Affinity Ligands and Their Role In Site-specific Conjugation Of Unmodified Antibodiesmentioning

confidence: 99%

Affinity-Based Methods for Site-Specific Conjugation of Antibodies

2021

Self Cite

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Conjugation of various reagents to antibodies has long been an elegant way to combine the superior binding features of the antibody with other desired but non-natural functions. Applications range from labels for detection in different analytical assays to the creation of new drugs by conjugation to molecules which improves the pharmaceutical effect. In many of these applications, it has been proven advantageous to control both the site and the stoichiometry of the conjugation to achieve a homogeneous product with predictable, and often also improved, characteristics. For this purpose, many research groups have, during the latest decade, reported novel methods and techniques, based on small molecules, peptides, and proteins with inherent affinity for the antibody, for site-specific conjugation of antibodies. This review provides a comprehensive overview of these methods and their applications and also describes a historical perspective of the field.

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“…reported a similar method, by using BPA incorporated into the Z domain, based on site‐specific labeling to immobilize mAbs on nanoparticles . The field of photoaffinity labeling of native antibodies is now widespread, and recently Park et al . and Vance et al .…”

Section: Affinity Peptide Labeling For Site‐specific Conjugation Of Nmentioning

confidence: 99%

Recent Chemical Approaches for Site‐Specific Conjugation of Native Antibodies: Technologies toward Next‐Generation Antibody–Drug Conjugates

Yamada

Ito

2019

ChemBioChem

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Antibody–drug conjugates (ADCs), which consist of three components, antibody, linker, and payload, can function as “magic bullets”. These conjugates offer the ability to target drug delivery to specific cells, based on cell‐specific recognition and the binding of an antigen by a monoclonal antibody (mAb). In particular, by delivering a cytotoxic payload to cancer cells, ADCs are expected to provide a breakthrough in oncology treatments by providing a way to increase efficacy and decrease toxicity, in comparison with traditional chemotherapeutic treatments. The development of ADC therapeutics has dramatically progressed in the past decade and two ADCs have been approved and used as anticancer drugs in the clinic. However, several critical issues regarding the performance of ADCs are still being discussed and investigated. Indeed, in the past few years, several groups have reported that, changing the number and position of the drug payloads in the ADCs, affects the pharmacokinetics, drug release rates, and biological activity. The use of conventional heterogeneous conjugation methods for ADC preparation results in the drug/antibody ratio and connecting position of the payload having stochastic distributions. Therefore, it is important to investigate how these potential problems can be circumvented through site‐specific conjugation. Herein, various site‐specific chemical conjugation strategies with native mAbs that are currently used for the production of ADCs, including residue‐selective labeling for generating ADCs, disulfide rebridging, and affinity‐peptide‐mediated site‐specific chemical conjugation technologies, are reviewed and described.

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In vivo biotinylation and incorporation of a photo‐inducible unnatural amino acid to an antibody‐binding domain improve site‐specific labeling of antibodies

Cited by 20 publications

References 29 publications

High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody‐Binding Domains

High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody‐Binding Domains

Affinity-Based Methods for Site-Specific Conjugation of Antibodies

Recent Chemical Approaches for Site‐Specific Conjugation of Native Antibodies: Technologies toward Next‐Generation Antibody–Drug Conjugates

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