A homogeneous fluorescence-based method to measure antibody internalization in tumor cells

Gong, Haibiao; Urlacher, Teresa M.

doi:10.1016/j.ab.2014.09.008

Cited by 9 publications

(3 citation statements)

References 12 publications

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“…Conversely, IRDye QC-1 (QC-1; Li-Cor, Inc.) is a dark quencher with a much broader absorbance spectrum that encompasses the NIR and visible range 36 , 37 . To date, QC-1 has been used as a quencher in activatable probes in imaging of antibody internalization 38 , 39 , enzymatic activity 40 , and as a marker for photoacoustic imaging 41 - 43 . Yet, the characterization of the binding of QC-1- conjugated protein ligands to their respective receptors in cell-based assays as well as of the QC-1 behavior as an acceptor in FLI-FRET applications in vitro or in vivo is severely lacking.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor

et al. 2020

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Rationale: Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in live intact animals, which is critical to assess the delivery efficacy of therapeutics. However, to date, non-invasive imaging approaches that can simultaneously measure cellular drug delivery efficacy and metabolic response are lacking. A major challenge for the implementation of concurrent optical and MFLI-FRET in vivo whole-body preclinical imaging is the spectral crowding and cross-contamination between fluorescent probes. Methods: We report on a strategy that relies on a dark quencher enabling simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. Several optical imaging approaches, such as in vitro NIR FLI microscopy (FLIM) and in vivo wide-field MFLI, were used to validate a novel donor-dark quencher FRET pair. IRDye 800CW 2-deoxyglucose (2-DG) imaging was multiplexed with MFLI-FRET of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) to monitor tumor metabolism and probe uptake in breast tumor xenografts in intact live nude mice. Immunohistochemistry was used to validate in vivo imaging results. Results: First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700). Second, we report on simultaneous in vivo imaging of the metabolic probe 2-DG and MFLI-FRET imaging of Tf-AF700/Tf-QC-1 uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery. Conclusions: Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

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Section: Introductionmentioning

confidence: 99%

Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor

et al. 2020

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“…However, these proteins bind only monovalently and do not stimulate endocytosis after binding. In contrast, IgGs are bivalent and many are established for enhanced cellular uptake after binding due to increased endocytosis through triggering receptor dimerization or oligomerization by their crosslinking ability 38,39 . For these reasons, IgGs appear more promising and more generally applicable.…”

Section: Discussionmentioning

confidence: 99%

Towards targeted Cas9 (CRISPR‐Cas) delivery: Preparation of IgG antibody‐Cas9 conjugates using a split intein

Pasch,

Bäumer,

Bäumer

et al. 2024

Journal of Peptide Science

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The CRISPR‐Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease‐protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9‐RNP. We opted for protein trans‐splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine‐less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG‐Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double‐strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N‐terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG‐Cas9 provides the first step towards a novel CRISPR‐Cas9 targeting approach involving the preformed RNP complex.

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“…Conversely, IRDye QC-1 (QC-1; Li-Cor, Inc.) is a recently developed dark quencher with a much broader absorbance spectrum that encompasses the NIR and visible range. 36,37 To date, QC-1 has been used as a quencher in activatable probes in imaging of antibody internalization, 38,39 enzymatic activity, 40 and as a marker for photoacoustic imaging. 41,42 To the best of our knowledge, this is the first report of QC-1 complete characterization as an acceptor for NIR FLI-FRET in vivo imaging in live intact animals.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Non-invasive in vivo Imaging to Assess Metabolism and Receptor Engagement in Tumor Xenografts

Rudkouskaya

Sinsuebphon

Ochoa

et al. 2019

Preprint

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Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. To date, non-invasive quantitative imaging modalities that can comprehensively assess simultaneous cellular drug delivery efficacy and therapeutic response are lacking. In this regard, Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in vivo and subsequent intracellular internalization, which is critical to assess the delivery efficacy of targeted therapeutics. However, implementation of multiplexing optical imaging with FRET in vivo is challenging to achieve due to spectral crowding and cross-contamination. Herein, we report on a strategy that relies on a dark quencher that enables simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700) using in vitro NIR FLI microscopy and in vivo wide-field MFLI imaging. Second, we report on simultaneous in vivo imaging of the metabolic probe IRDye 800CW 2-deoxyglucose (2-DG) and MFLI-FRET imaging of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery. Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

show abstract

A homogeneous fluorescence-based method to measure antibody internalization in tumor cells

Cited by 9 publications

References 12 publications

Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor

Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor

Towards targeted Cas9 (CRISPR‐Cas) delivery: Preparation of IgG antibody‐Cas9 conjugates using a split intein

Multiplexed Non-invasive in vivo Imaging to Assess Metabolism and Receptor Engagement in Tumor Xenografts

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