Antibodies with integrated endosome escape and multi-directional intracellular trafficking-control capabilities for molecular transport and accumulation of a BODIPY-based dye
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".