A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

Yamashita, Makiko; Kitano, Shigehisa; Aikawa, Hiroaki; Kuchiba, Aya; Hayashi, Mitsuhiro; Yamamoto, Noboru; Takagi, Kenji; Hamada, Akinobu

doi:10.1038/srep19772

Cited by 50 publications

(34 citation statements)

References 27 publications

(31 reference statements)

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“…2B) . The observed level of direct target killing by E8 in-vitro and ex-vivo ranged between~20-40% in independent biological replicate experiments, which is comparable to the levels observed with approved anti-cancer biologicals [27][28][29] . E8 demonstrated remarkable selectivity at the target cell level, killing TNBC9 but not MCF10A cells, which were used as negative targets in the in-vitro evolution process (Fig.…”

Section: Resultssupporting

confidence: 64%

Discovery of tumoricidal DNA oligonucleotides by effect-directedin-vitroevolution

Mamet

Amir

Lavi

et al. 2019

Preprint

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Our current model of drug discovery is challenged by the relative ineffectiveness of drugs against highly variable and rapidly evolving diseases and their relatively high incidence of adverse effects due to poor selectivity. Here we describe a robust and reproducible platform which could potentially address these limitations. The platform enables rapid, de-novo discovery of DNA aptamers evolved in-vitro to exert specific biological effects on target cells. Unlike conventional aptamers, which are selected by their ligand binding capacity, this platform is driven directly by therapeutic effect and selectivity towards target vs negative target cells. The process could, therefore, operate without any a-priori knowledge (e.g. mutations, biomarker expression, or known drug resistance) of the target. We report the discovery of DNA aptamers with direct and selective cytotoxicity towards several tumor cell lines as well as primary, patient-derived solid and hematological tumors, some with chemotherapy resistance. Aptamers discovered by this platform exhibited favorable biodistribution in animals, persistence in target tumors up to 48 hours after injection, and safety in human blood. These aptamers showed remarkable efficacy in-vivo as well as ex-vivo in freshly obtained, 3D cultured human tumors resistant to multiple chemotherapies. With further improvement, these findings could lead to a drug discovery model which is target-tailored, mechanism-flexible, and nearly on-demand.

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Section: Resultssupporting

confidence: 64%

Discovery of tumoricidal DNA oligonucleotides by effect-directedin-vitroevolution

Mamet

Amir

Lavi

et al. 2019

Preprint

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“…[6][7][8][9][10][11][12]. The observed level of direct target killing by E8 in vitro and ex vivo ranged between~20-40% in independent biological replicate experiments, which is comparable to the levels observed with approved anti-cancer biologicals [27][28][29] . E8 demonstrated remarkable selectivity at the target cell level, killing TNBC9 but not MCF10A cells, which were used as negative targets in the in vitro evolution process ( Fig.…”

Section: Resultsmentioning

confidence: 79%

Discovery of tumoricidal DNA oligonucleotides by response-directed in vitro evolution

et al. 2020

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Drug discovery is challenged by ineffectiveness of drugs against variable and evolving diseases, and adverse effects due to poor selectivity. We describe a robust platform which potentially addresses these limitations. The platform enables rapid discovery of DNA oligonucleotides evolved in vitro for exerting specific and selective biological responses in target cells. The process operates without a priori target knowledge (mutations, biomarkers, etc). We report the discovery of oligonucleotides with direct, selective cytotoxicity towards cell lines, as well as patient-derived solid and hematological tumors. A specific oligonucleotide termed E8, induced selective apoptosis in triple-negative breast cancer (TNBC) cells. Polyethylene glycol-modified E8 exhibited favorable biodistribution in animals, persisting in tumors up to 48-hours after injection. E8 inhibited tumors by 50% within 10 days of treatment in patient-derived xenograft mice, and was effective in ex vivo organ cultures from chemotherapy-resistant TNBC patients. These findings highlight a drug discovery model which is target-tailored and on-demand.

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“…In spite of spontaneous release, the intracellular fluorescence of the more stable CFSE label still can be sufficient for discriminating labeled target cells from nonlabeled effector cells by flow cytometry even 4 h after labeling. In fact, detection and quantification of dead and live CFSE‐labeled target cells using flow cytometry was shown to be more sensitive than release assays . The spectral separation also allows the separate observation of PI uptake by target cells with damaged cell membrane.…”

Section: Resultsmentioning

confidence: 99%

“…While this latter was originally regarded as reproducible and easy‐to‐perform, several of its drawbacks have been revealed lately. It turned out that it is semi‐quantitative, only moderately sensitive, prone to poor labeling, and there is high spontaneous release from some target cell lines . In addition, the 51 Cr release assay raises the obvious biohazard and disposal issues, and all these contraindications have led to attempts for its replacement with nonradioactive assays .…”

mentioning

confidence: 99%

Quantitating ADCC against adherent cells: Impedance‐based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response

2017

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Running headline (45 chars. incl. space) Impedance-based cell analyzer for ADCC Conflicts of interest:None of the authors have conflicts interest. AbstractMonoclonal antibody-based immunotherapeutics will dominate Pharma's next generation of blockbuster drugs, and Fc-associated functions, including antibody dependent cellular cytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies. Therefore, quantitative evaluation of ADCC is required during drug development. Our objective was to find the most suitable and reliable non-radioactive method for quantitative analysis of in vitro ADCC against adherent cells, which often serve as models for solid tumors. The test system was comprised the HER2 positive JIMT-1 cells targeted by the specific therapeutic antibodies trastuzumab (Herceptin ® ) and pertuzumab (Perjeta ® ). These cells are resistant to the direct biological effects of these antibodies, and therefore allow the isolated assessment of ADCC. We compared fluorescein diacetate (FDA) and carboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternative to 51 Cr release; propidium iodide (PI) uptake revealing increased membrane permeability; the PanToxiLux assay measuring ADCC induced proapoptotic protease activity in flow cytometry; and an impedance-based real time cell adhesion test. We found that release assays are compromised by high spontaneous release of the label. PI uptake could not differentiate well between spontaneous NK activity and specific ADCC. The PanToxiLux assay, besides allowing for shorter assay times, offers improvement over the previous approaches in distinguishing spontaneous and antibody mediated NK action, but, probably owed to the prolonged detached state of adherent target cells, only at highly saturating antibody concentrations. In the case of adherent target cells, impedance-based cell analysis attains functional information exclusively on the target cells without having to label them for distinguishing from effectors or assay readout. It also allows continuous monitoring for days, and specifically detects target cell detachment, as the final functional consequence of ADCC. The sensitivity of this method even allows for quantitating the additivity and saturability of ADCC as a function of antibody concentration. We conclude that impedance-based assays are the most sensitive for quantitatively assessing in vitro ADCC on adherent target cells.

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A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

Cited by 50 publications

References 27 publications

Discovery of tumoricidal DNA oligonucleotides by effect-directedin-vitroevolution

Discovery of tumoricidal DNA oligonucleotides by effect-directedin-vitroevolution

Discovery of tumoricidal DNA oligonucleotides by response-directed in vitro evolution

Quantitating ADCC against adherent cells: Impedance‐based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response

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