A Cell-Based Reporter Assay Measuring the Activation of Fc Gamma Receptors Induced by Therapeutic Monoclonal Antibodies

Aoyama, Michihiko; Tada, Minoru; Ishii‐Watabe, Akiko

doi:10.1007/978-1-4939-8958-4_21

Cited by 5 publications

(5 citation statements)

References 16 publications

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“…Therefore, we focused on the relationship between FcγRs and cytotoxicity of ADC aggregates to unravel the mechanism by which the cytotoxicity of ADCs induced by aggregation was enhanced. To evaluate the FcγR-activation properties of ADC aggregates, we performed the reporter assays using Jurkat/FcγRs/NFAT-Luc reporter cell lines which we previously developed ( 14 , 15 ). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…SK-BR-3 cells (an HER2 + human breast cancer cell line, ATCC® HTB-30) was obtained from ATCC. Jurkat cells expressing human FcγRIIa or FcγRIIIa with the Nuclear Factor of Activated T cells (NFAT)-driven luciferase reporter (Jurkat/FcγRIIa/NFAT-Luc, Jurkat/ FcγRIIIa/NFAT-Luc) were established previously ( 14 , 15 ). TMNK-1 cells were maintained in Medium 200 (Thermo Fisher Scientific, Waltham, MA) supplemented with Low Serum Growth Supplement (Thermo Fisher Scientific) at 37°C in a humidified atmosphere containing 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

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Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates

et al. 2021

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Purpose Antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) conjugated with highly toxic payloads, achieve high tumor killing efficacy due to the specific delivery of payloads in accordance with mAbs’ function. On the other hand, the conjugation of payloads often increases the hydrophobicity of mAbs, resulting in reduced stability and increased aggregation. It is considered that mAb aggregates have potential risk for activating Fcγ receptors (FcγRs) on immune cells, and are internalized into cells via FcγRs. Based on the mechanism of action of ADCs, the internalization of ADCs into target-negative cells may cause the off-target toxicity. However, the impacts of aggregation on the safety of ADCs including off-target cytotoxicity have been unclear. In this study, we investigated the cytotoxicity of ADC aggregates in target-negative cells. Methods The ADC aggregates were generated by stirring stress or thermal stress. The off-target cytotoxicity of ADC aggregates was evaluated in several target-negative cell lines, and FcγR-activation properties of ADC aggregates were characterized using a reporter cell assay. Results Aggregation of ADCs enhanced the off-target cytotoxicity in several target-negative cell lines compared with non-stressed ADCs. Notably, ADC aggregates with FcγR-activation properties showed dramatically enhanced cytotoxicity in FcγR-expressing cells. The FcγR-mediated off-target cytotoxicity of ADC aggregates was reduced by using a FcγR-blocking antibody or Fc-engineering for silencing Fc-mediated effector functions. Conclusions These results indicated that FcγRs play an important role for internalization of ADC aggregates into non-target cells, and the aggregation of ADCs increases the potential risk for off-target toxicity.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates

et al. 2021

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“…We previously developed FcgR-expressing reporter cell lines in which activation of FcgRs led to the expression of luciferase reporter and reported that these reporter cells could be useful for the evaluation of Fc-mediated immune cell activation by mAbs. [21][22][23] In this study, we used the FcgR-expressing reporter cell lines to examine the FcgR-activation properties of mAb aggregates with different characteristics, and we revealed that FcgR activation by mAb aggregates depends on the Fc function of the native (nonaggregated) mAbs. We also showed that mAb aggregates smaller than 1 mm can directly activate FcgRs.…”

Section: Introductionmentioning

confidence: 99%

Fcγ Receptor Activation by Human Monoclonal Antibody Aggregates

Tada

Aoyama

Ishii‐Watabe

2020

Journal of Pharmaceutical Sciences

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Protein aggregates are a potential risk factor for immunogenicity. The measurement, characterization, and control of protein aggregates in drug products are indispensable for the development of biopharmaceuticals, including therapeutic mAbs. In this study, Fcg receptor (FcgR)-expressing reporter cell lines were used to analyze the FcgR-activation properties of mAb aggregates. Comparison of aggregates of mAbs harboring different IgG subclasses revealed that the FcgR-activation profiles of the mAb aggregates were dependent on IgG subclass. In addition, aggregates of Fc-engineered mAb with enhanced FcgRactivation properties exhibited stronger activation of FcgRs than was observed in the wild-type aggregates, whereas aggregates of Fc-engineered mAb with decreased FcgR-activation properties showed reduced activation. These results suggest that FcgR activation by mAb aggregates depends greatly on the Fc functions of the native (nonaggregated) mAbs. We also showed that aggregates of mAbs smaller than 1 mm in size have the potential to directly activate FcgRs. Unintended immune cell activation can be induced on account of FcgR activation by mAb aggregates and such FcgR activation may contribute to immunogenicity, and therefore, analysis of the FcgR-activation properties of mAb aggregates using FcgR-expressing reporter cell lines is a promising approach for the characterization of mAb aggregates.

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“…Our methodology provides a comprehensive system, supporting the assessment of essentially all FcγRs, which presents an advantage over previously developed sIC detection and FcγR activation assays (Cheng et al , 2014; Tada et al , 2014; Szittner et al , 2016; Hsieh et al , 2017; Stopforth et al , 2018; Aoyama et al , 2019). In contrast to currently available commercial assays, detecting sICs by C1q‐CIC or C3d ELISA in the micromolar range, our assay measures overall sIC bioactivity in the nanomolar range and has a sole specificity for IgG sICs.…”

Section: Discussionmentioning

confidence: 88%

Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

Chen¹,

Maul-Pavicic

Holzer

et al. 2021

EMBO Mol Med

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Fc-gamma receptor (FccR) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. We developed a comprehensive reporter cell panel detecting activation of FccRs. The reporter cell lines were integrated into an assay that enables the quantification of sIC reactivity via ELISA or a faster detection using flow cytometry. This identified FccRIIA(H) and FccRIIIA as the most sIC-sensitive FccRs in our test system. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size reproducing for the first time a complete Heidelberger-Kendall curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent FccR activation has predictive capabilities regarding severity of SLE disease. The assay provides a sensitive and scalable tool to evaluate the size, amount, and bioactivity of sICs in all settings.

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A Cell-Based Reporter Assay Measuring the Activation of Fc Gamma Receptors Induced by Therapeutic Monoclonal Antibodies

Cited by 5 publications

References 16 publications

Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates

Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates

Fcγ Receptor Activation by Human Monoclonal Antibody Aggregates

Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

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