A Flow Cytometry-Based Assay for the Evaluation of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) in Cancer Cells

Salinas-Jazmín, Nohemí; Hisaki-Itaya, Emiliano; Velasco-Velázquez, Marco A.

doi:10.1007/978-1-4939-0856-1_16

Cited by 11 publications

(10 citation statements)

References 9 publications

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“…The recent confirmation of the clinical efficacies of several immunotherapeutic drugs in patients with cancers has promoted the development of this treatment strategy. In particular, the use of monoclonal antibodies (mAbs) for cancer therapy is one of the most successful and important strategies for treating cancer patients 1 . Such mAbs can kill tumor cells by (1) blocking the function of the target molecule, (2) mediating the delivery of cytotoxic drugs, (3) affecting the tumor vasculature or stroma, and/or (4) triggering immune-mediated cell killing mechanisms.…”

mentioning

confidence: 99%

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

Yamashita¹,

Kitano

Aikawa³

et al. 2016

Sci Rep

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Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The 51Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies.

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confidence: 99%

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

Yamashita¹,

Kitano

Aikawa³

et al. 2016

Sci Rep

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“…As one of the major antibody effector functions against tumor cells 2 , we next determined whether our chimeric anti-EBV TCR-like mAbs could induce ADCC of EBV BLCLs. CFSE-labeled BLCLs were incubated with the respective antibodies plus PBMCs, and ADCC was evaluated via flow cytometry 32 (Supplementary Figure S4 ). E1, L1 and L2 exhibited significantly enhanced cellular cytotoxicity against EBV BLCLs of various HLA-A*02 subtypes including A*02:01 A*02:03 WGP6, A*02:06 CM371 and A*02:03 A*02:07 CM956.…”

Section: Resultsmentioning

confidence: 99%

TCR–like antibodies mediate complement and antibody-dependent cellular cytotoxicity against Epstein-Barr virus–transformed B lymphoblastoid cells expressing different HLA-A*02 microvariants

Lai

Choo

Tan

et al. 2017

Sci Rep

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Epstein-Barr virus (EBV) is a common gammaherpesvirus associated with various human malignancies. Antibodies with T cell receptor-like specificities (TCR-like mAbs) provide a means to target intracellular tumor- or virus-associated antigens by recognising their processed peptides presented on major histocompatibility complex (MHC) class I (pMHC) complexes. These antibodies are however thought to be relevant only for a single HLA allele. Here, we show that HLA-A*02:01-restricted EBV antigenic peptides EBNA1562-570, LMP1125-133 and LMP2A426-434 display binding degeneracy towards HLA-A*02 allelic microvariants, and that these pMHC complexes are recognised by anti-EBV TCR-like mAbs E1, L1 and L2 raised in the context of HLA-A*02:01. These antibodies bound endogenously derived pMHC targets on EBV–transformed human B lymphoblastoid cell lines expressing A*02:01, A*02:03, A*02:06 and A*02:07 alleles. More importantly, these TCR-like mAbs mediated both complement-dependent and antibody-dependent cellular cytotoxicity of these cell lines in vitro. This finding suggests the utility of TCR-like mAbs against target cells of closely related HLA subtypes, and the potential applicability of similar reagents within populations of diverse HLA-A*02 alleles.

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“…Monoclonal antibody‐based immunotherapeutics will dominate Pharma's next generation of blockbuster drugs . The evaluation of the biological activity of mAbs is required during most drug development , and the European Medicines Agency and the U.S. Food and Drug Administration have each drafted guidelines for the development of biosimilar products; extensive structural and functional characterization of the proposed product is recommended.…”

mentioning

confidence: 99%

“…Trastuzumab (Herceptin ® ) and pertuzumab (Perjeta ® ) are both humanized monoclonal antibodies in clinical use targeting distinct extracellular regions of HER2 . Their mechanisms of action include direct biological effects as well , but ADCC appears to be of the greatest clinical relevance , and recently it was established that antibody doses exceeding the highest approved clinical doses do not saturate the ADCC response . This emphasizes the need for quantitatively analyzing the dose dependence of ADCC for antibodies under development or evaluation.…”

mentioning

confidence: 99%

“…JIMT‐1 cells, even though they express the target HER2 molecule, have proved to be in vitro resistant to the direct cell biological effects of both trastuzumab and pertuzumab. However, both in vitro, and when freshly inoculated into SCID mice, JIMT‐1 tumors are sensitive to ADCC mediated by these antibodies . Therefore, their use allows the isolated observation and quantification of ADCC without being disturbed by the direct biological effects of these antibodies.…”

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confidence: 99%

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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 Flow Cytometry-Based Assay for the Evaluation of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) in Cancer Cells

Cited by 11 publications

References 9 publications

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

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

TCR–like antibodies mediate complement and antibody-dependent cellular cytotoxicity against Epstein-Barr virus–transformed B lymphoblastoid cells expressing different HLA-A*02 microvariants

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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