Validation of an ADCC assay using human primary natural killer cells to evaluate biotherapeutic products bearing an Fc region

González‐González, Edith; Camacho-Sandoval, Rosa; Jiménez-Uribe, Alexis Paulina; Montes-Luna, Alejandra; Cortés-Paniagua, Ilselena; Sánchez-Morales, Jazmín; Muñoz-García, Leslie; Tenorio-Calvo, Alejandra; López‐Morales, Carlos A.; Velasco-Velázquez, Marco A.; Pavón, Lenin; Pérez‐Tapia, Sonia Mayra; Medina‐Rivero, Emilio

doi:10.1016/j.jim.2018.11.002

Cited by 5 publications

(3 citation statements)

References 21 publications

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“…Each of these methods utilizes fluorescent molecules to distinguish target cells from effector cells and to measure cell viability as indicator of efficiency of tumor killing. Several other groups have recently published variations on these experimental protocols (Alrubayyi et al, 2018;Carter et al, 2019;González-González et al, 2019;Kamen et al, 2019;Tanaka et al, 2019;van der Haar Àvila, Marmol, Cany, Kiessling, & Pico de Coaña, 2019;Yamashita et al, 2016). These techniques have several advantages over other methods, including the ability to simultaneously measure the amount of target antibody bound to the cells and cell viability.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions

Miller

Finn

2020

Methods in Enzymology

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Monoclonal antibody-based therapies are increasingly being used to treat cancer. Some mediate their therapeutic effects through modifying the function of immune cells globally, while others bind directly to tumor cells and can recruit immune effector cells through their Fc regions. As new direct-binding agents are developed, having the ability to test their Fc-mediated functions in a high-throughput manner is important for selecting antibodies with immune effector properties. Here, using monoclonal anti-CD20 antibody (rituximab) as an example and the CD20 + Raji cell line as tumor target, we describe flow cytometry-based assays for determining an antibody's capacity for mediating antibody-dependent cellular cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). These assays are sensitive, reliable, affordable and avoid the use of radioactivity.

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Section: Discussionmentioning

confidence: 99%

Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions

Miller

Finn

2020

Methods in Enzymology

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“…ADCC activity of the HLA-A2 mAbs were assessed by a plate-based assay using an adaptation of a method described previously. 116 Briefly, A375 cells which expressed GFP, obtained by transducing A375 cells (ATCC®, USA) with Ppy RE9-GFP retroviral backbone 117 (50,000 cells/well) were added to the titrated HLA-A2 mAbs in a total of 100 μL of complete assay medium (RPMI-1640 media (Corning, USA) supplemented with 10% heat-inactivated FBS (Biowest), 1 mM sodium pyruvate (Corning, USA), 1X MEM nonessential amino acids (Corning, USA) and 1X Penicillin Streptomycin (Corning, USA)), in a sterile, U-bottom 96 well suspension culture plate (Greiner Bio-one). The plate was then incubated at 37°C at 5% CO 2 for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Afucosylation of HLA-specific IgG1 as a potential predictor of antibody pathogenicity in kidney transplantation

Bharadwaj

Shrestha

Pongrácz

et al. 2022

Cell Reports Medicine

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“…The assay was performed as previously described with modifications 40 , 41 . PBMC stimulation was performed in Immulon II plates coated with relevant antigens as described above, at 50,000 cells/well.…”

Section: Methodsmentioning

confidence: 99%

Development of antibody-dependent cellular cytotoxicity in response to recombinant and live-attenuated herpes zoster vaccines

et al. 2022

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Zoster vaccines generate antibody responses against varicella-zoster virus (VZV). We compared antibody-dependent cell cytotoxicity (ADCC) elicited by zoster vaccine live (ZVL) and recombinant zoster vaccine (RZV). ADCC mediated by antibodies against VZV lysate (VZV-ADCC) and recombinant glycoprotein E (gE-ADCC) was measured using plasma from 20 RZV- and 20 ZVL-recipients, including half 50–60-years-old and half ≥70-years-old. Solid phase-bound anti-VZV antibodies stimulated TNFα in NK cells as measured by flow cytometry or ELISA. VZV-ADCC pre- and post-immunization was higher in younger vaccinees. ZVL did not appreciably increase VZV-ADCC, whereas RZV increased VZV-ADCC in older vaccinees. ELISA-measured gE-ADCC was similar across groups pre-immunization; significantly increased after ZVL; and RZV and was higher in younger RZV than ZVL recipients. IgG3 antibodies increased after RZV and ZVL, with greater anti-gE than anti-VZV responses. Moreover, gE-ADCC strongly correlated with anti-gE antibody avidity, but there were no appreciable correlations between VZV-ADCC and avidity. NK cells stimulated by anti-gE antibodies showed increased IFNγ and CD107a expression, which was not observed with anti-VZV antibodies. In conclusion, anti-gE antibodies generated more robust ADCC than anti-VZV antibodies. RZV induced higher ADCC antibodies than ZVL depending on the antigen and age of vaccinees. Older adults had lower ADCC antibodies before and after vaccination than younger adults.

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Validation of an ADCC assay using human primary natural killer cells to evaluate biotherapeutic products bearing an Fc region

Cited by 5 publications

References 21 publications

Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions

Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions

Afucosylation of HLA-specific IgG1 as a potential predictor of antibody pathogenicity in kidney transplantation

Development of antibody-dependent cellular cytotoxicity in response to recombinant and live-attenuated herpes zoster vaccines

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