Antibodies with integrated endosome escape and multi-directional intracellular trafficking-control capabilities for molecular transport and accumulation of a BODIPY-based dye
Notice bibliographique
Résumé
1215 Objectives The understanding of normal and aberrant cellular physiological processes is increasingly dependent on the ability to visualize and quantify specific cells of interest with high spatial resolution. Targeting cells with monoclonal antibodies (mAbs) tagged with fluorescent probes, herein termed antibody-conjugates (ACs) has been a mainstay for cell surface phenotype interrogation. Previous attempts have exploited cellular physiology to provide ACs with a level of subcellular directional control in order to visualize intracellular events. The current mechanistic center point for ACs to effectively accumulate fluorescent probes in target cells is perceived to be acidic pH-activated or protease-activatable AC degradation in the lysosome. Unfortunately this process is ineffective as multi-drug resistant transporters efficiently export liberated probes. ACs can also be recycled back outside the cell. Our goal is to develop a versatile technology that addresses these limitations to improve fluorescent probe intracellular delivery. We present the bile acid, cholic acid (ChAc) linked to short inert (i.e. does not penetrate the plasma membrane) peptides that harbour a nuclear localization signal (NLS). We exploit the ChAc to selectively disrupt endosome membranes and the NLS to accumulate inside the nucleus. We evaluate the generalization of ChAcNLS on different antibodies. Methods MAbs 7G3 and 6G7, specific for IL-3Rα and mAb A14, specific for IL-5Rα were conjugated with ChAcNLS and control peptides using the crosslinker sulfo-SMCC. SDS-PAGE was used to determine peptide-to-Ab ratio. IL-3Rα+ and IL-3Rα- cancer cells, were treated with 200nM ACs for 1h at 37C followed by trypsinization and cellular fractionation. Fluorescently-tagged secondary antibodies were used and cells or isolated nuclei assessed by confocal microscopy and flow cytometry. AC internalization and antigen recycling was also evaluated with and without pretreatment of the cholesterol inhibitor filipin. The fluorescent dye 4,4-difluoro-4-bora-3a,4a, diaza-s-indacene (BODIPY) carboxy derivative (cBPY) was prepared in-house and attached as the molecular cargo. Fluorescence spectrum of cBPY were determined by a fluorescence microplate reader. cBPY was conjugated to ACs using the crosslinker EDC and quantification of cBPY per AC evaluated by fluorescence-to-protein absorbance ratio experiments. HT-1376 muscle invasive bladder cancer (MIBC) cells expressing IL-5Rα were treated with 400nM cBPY-A14-ChAcNLS. The delivery of cBPY to the nucleus and its affect on cellular fluorescence was evaluated by live confocal laser microscopy. Results ChAcNLS-AC could increase their nuclear and intracellular accumulation by at least 5-fold relative to non-ChAcNLS-modified ACs. ChAcNLS-ACs maintained target cell selectivity. Interestingly, fillipin treatment blocked receptor mediated internalization of ChAcNLS-mAbs, which suggests internalization is dependent on intact lipid rafts. Importantly, ChAcNLS-ACs also selectively increased the intracellular and nuclear transportation of cBY in target MIBC cells by a factor greater than 9 over traditional ACs. Conclusions Our novel AC accumulation technology (ChAcNLS) is generalizable to the mAbs evaluated in this study and enhanced the nuclear accumulation of cBPY to increase specific-fluorescent signals from within selected subcellular locations in target cells. Thus ChAcNLS could have tremendous applications in the fields of life science and oncology.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,001 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».