Simultaneous and Independent Dual Site-Specific Self-Labeling of Recombinant Antibodies

Wollschlaeger, Carolin; Meinhold‐Heerlein, Ivo; Cong, Xiaojing; Bräutigam, Karen; Fiore, Stefano; Zeppernick, Felix; Klockenbring, Torsten; Stickeler, Elmar; Barth, Stefan; Hussain, Ahmad Fawzi

doi:10.1021/acs.bioconjchem.8b00545

Cited by 8 publications

(7 citation statements)

References 49 publications

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“…Others have described strategies to introduce two cargos site-specifically on a single protein by introducing a combination of tags recognized by orthogonal enzymes, self-labelling tags, engineered residues or inteins [39][40][41][42][43][44] . All these strategies have their advantages, but share the requirement to know the sequence of the antibody's variable region to accommodate recombinant expression.…”

Section: Discussionmentioning

confidence: 99%

Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

Gall

Schoot

Ramos-Tomillero

et al. 2020

Preprint

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I.AbstractFunctionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theragnostics, and antibody-drug conjugates (ADC). Antibody functionalization is classically achieved by coupling payloads onto lysine or cysteine residues. However, such stochastic strategies typically lead to heterogenous products, bearing a varying number of payloads. This affects bioconjugate efficacy and stability, as well as its in vivo biodistribution, and therapeutic index, while potentially obstructing the binding sites and leading to off-target toxicity. In addition, therapeutic and theragnostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labelling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogenous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab’ fragments bearing two distinct site-specific labelling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab’ molecule (DTFab’), which can be easily isolated in high yields. To demonstrate feasibility, we functionalized the DTFab’ with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theragnostics, and next-generation ADCs.

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Section: Discussionmentioning

confidence: 99%

Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

Gall

Schoot

Ramos-Tomillero

et al. 2020

Preprint

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“…The commercial product is named SNAP-tag, which is a monomeric 20 kDa hAGT mutant that takes up O 6 -benzylguanine (BG) derivatives as substrates (Figure b). , The CLIP-tag, which is mutated from SNAP-tag that reacts exclusively with O 2 -benzylcytosine (BC) derivatives, was published a few years after the SNAP-tag . SNAP- and CLIP-tags have orthogonal substrate specificities, thus enabling the simultaneous labeling reactions with little cross-reactivity. − …”

Section: Introductionmentioning

confidence: 99%

SNAP/CLIP-Tags and Strain-Promoted Azide–Alkyne Cycloaddition (SPAAC)/Inverse Electron Demand Diels–Alder (IEDDA) for Intracellular Orthogonal/Bioorthogonal Labeling

Macias-Contreras

Little

et al. 2020

Bioconjugate Chem.

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Labeling a protein of interest (POI) with a fluorescent reporter is a powerful strategy for studying protein structures and dynamics in their native environments. Compared to fluorescent proteins, synthetic dyes provide more choices in photophysical or photochemical attributes to microscopic characterizations. The specificity of bioorthogonal reactions in conjunction with the fidelity of subcellular destinations of genetically encoded protein tags can be employed to label POIs in live and fixed cells in a two-step process. In the present study the orthogonality of the strain-promoted azide–alkyne cycloaddition (SPAAC) and the inverse electron demand Diels–Alder (IEDDA) reaction is corroborated in concurrent labeling of two different intracellular targets. An azido group and a strained alkene are first installed at specific subcellular locations via orthogonal enzymatic reactions of the genetically incorporated SNAP- and CLIP-tags. The subsequent bioorthogonal reactions with fluorophores carrying matching reactive functionalities result in simultaneous dual labeling. The two-step “orthogonal-bioorthogonal” labeling process would increase the utilities of SNAP/CLIP-tags and, as a consequence, would expand the capability of decorating biological specimens with functionalities beyond fluorophores to potentially include spin labels, radioactive tracers, or catalysts.

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“…Moreover, the simultaneous incorporation of multiple distinct ncAAs of PTMs into one protein is desirable. As noted above, for simultaneous site-specific incorporation of several ncAAs into proteins, the dual genetic code expansion strategy has been applied to design two different stop codons or one stop codon together with one four-base codon (Hoesl and Budisa, 2011;Rashidian et al, 2013;Zang et al, 2015;Luo et al, 2016;Venkat et al, 2018;Wollschlaeger et al, 2018;Zheng et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Multiple Site-Specific One-Pot Synthesis of Two Proteins by the Bio-Orthogonal Flexizyme System

Xiao

Liu

Zhao

et al. 2020

Front. Bioeng. Biotechnol.

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Lysine acetylation is a reversible post-translational modification (PTM) vastly employed in many biological events, including regulating gene expression and dynamic transitions in chromatin remodeling. We have developed the first one-pot bio-orthogonal flexizyme system in which both acetyl-lysine (AcK) and non-hydrolysable thioacetyl-lysine (ThioAcK) were site-specifically incorporated into human histone H3 and H4 at different lysine positions in vitro, either individually or in pairs. In addition, the high accuracy of this system moving toward one-pot synthesis of desired histone variants is also reported.

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Simultaneous and Independent Dual Site-Specific Self-Labeling of Recombinant Antibodies

Cited by 8 publications

References 49 publications

Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

SNAP/CLIP-Tags and Strain-Promoted Azide–Alkyne Cycloaddition (SPAAC)/Inverse Electron Demand Diels–Alder (IEDDA) for Intracellular Orthogonal/Bioorthogonal Labeling

Multiple Site-Specific One-Pot Synthesis of Two Proteins by the Bio-Orthogonal Flexizyme System

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