Impact of Epidermal Growth Factor Receptor (EGFR) Cell Surface Expression Levels on Effector Mechanisms of EGFR Antibodies

Derer, Stefanie; Bauer, Philip; Lohse, Stefan; Scheel, Andreas H.; Berger, Sven; Kellner, Christian; Peipp, Matthias; Valerius, Thomas

doi:10.4049/jimmunol.1202037

Cited by 58 publications

(67 citation statements)

References 51 publications

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“…2C). CDC triggered by IgG1 Abs directed against EGFR has been recently demonstrated to depend on cell-surface expression levels of the Ag (9). To analyze whether the extent of CDC activity induced by anti-EGFR-IgG3 is controlled by EGFR expression levels, we quantified EGFR cell-surface expression levels by calibrated flow cytometry and correlated them to the extent of anti-EGFR-IgG3-triggered CML of the respective cell line.…”

Section: Cdc Is Induced By Anti-egfr-igg3 But Not By Anti-egfr-igg1mentioning

confidence: 99%

“…To analyze the effect of different EGFR expression levels on anti-EGFR-IgG1-or anti-EGFR-IgG3-mediated CDC, we used BHK-EGFR + #1-#5 cells, displaying varying expression levels of human EGFR (#1 , #2 , #3 , #4 , #5), in CDC assays (9). To exclude different EGFR binding capacities of anti-EGFR-IgG1 and anti-EGFR-IgG3 on these transfectants, we generated doseresponse curves for both Abs, as well as for the respective control Abs by flow cytometry analyses (Fig.…”

Section: Cdc Is Induced By Anti-egfr-igg3 But Not By Anti-egfr-igg1mentioning

confidence: 99%

“…Human epidermoid carcinoma cell line A431 (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and baby hamster kidney 21 (BHK-21) cells transfected with the human EGFR (9) were kept in RPMI 1640. The human colon carcinoma cell line DiFi and glioblastoma cell line A1207 (European Collection of Cell Cultures, Salisbury, U.K.) were kept in DMEM, and Chinese hamster ovarian K1 (CHO-K1) cells were kept in DMEM select according to manufacturer's instructions.…”

Section: Cell Linesmentioning

confidence: 99%

“…However, based on the finding that complement activation by Ig does not solely depend on C1q fixation (6), it might be hypothesized that other essential molecular features are required besides strong C1q binding for efficient induction of CDC, such as Ag cell-surface expression levels, the binding epitope of the Ab, Ags' mobility within the cell membrane, as well as Abs' isotype (7)(8)(9). This suggestion has been strengthened by results received from functional characterization of the therapeutic epidermal growth factor receptor (EGFR)-targeting Ab cetuximab, a mouse-human chimeric IgG1 molecule.…”

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confidence: 99%

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Epidermal Growth Factor Receptor Targeting IgG3 Triggers Complement-Mediated Lysis of Decay-Accelerating Factor Expressing Tumor Cells through the Alternative Pathway Amplification Loop

Rösner

Lohse

Peipp

et al. 2014

The Journal of Immunology

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Binding of C1q to target-bound IgG initiates complement-mediated lysis (CML) of pathogens, as well as of malignant or apoptotic cells, and thus constitutes an integral part of the innate immune system. Despite its prominent molecular flexibility and higher C1q binding affinity compared with human IgG1, IgG3 does not consistently promote superior CML. Hence the aim of this study was to investigate underlying molecular mechanisms of IgG1- and IgG3-driven complement activation using isotype variants of the therapeutic epidermal growth factor receptor (EGFR) Ab cetuximab. Both IgG1 and IgG3 Abs demonstrated similar EGFR binding and similar efficiency in Fab-mediated effector mechanisms. Whereas anti–EGFR-IgG1 did not promote CML of investigated target cells, anti–EGFR-IgG3 triggered significant CML of some, but not all tested cell lines. CML triggered by anti–EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement regulatory proteins CD55 and CD59, but not CD46. Notably, anti–EGFR-IgG3 promoted strong C1q and C3b, but relatively low C4b and C5b-9 deposition on analyzed cell lines. Furthermore, anti–EGFR-IgG3 triggered C4a release on all cells but failed to induce C3a and C5a release on CD55/CD59 highly expressing cells. RNA interference-induced knockdown or overexpression of membrane-bound complement regulatory proteins revealed CD55 expression to be a pivotal determinant of anti–EGFR-IgG3–triggered CML and to force a switch from classical complement pathway activation to C1q-dependent alternative pathway amplification. Together, these data suggest human anti–EGFR-IgG3, although highly reactive with C1q, to weakly promote assembly of the classical C3 convertase that is further suppressed in the presence of CD55, forcing human IgG3 to act mainly through the alternative pathway.

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Section: Cdc Is Induced By Anti-egfr-igg3 But Not By Anti-egfr-igg1mentioning

confidence: 99%

Section: Cdc Is Induced By Anti-egfr-igg3 But Not By Anti-egfr-igg1mentioning

confidence: 99%

Section: Cell Linesmentioning

confidence: 99%

mentioning

confidence: 99%

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Epidermal Growth Factor Receptor Targeting IgG3 Triggers Complement-Mediated Lysis of Decay-Accelerating Factor Expressing Tumor Cells through the Alternative Pathway Amplification Loop

Rösner

Lohse

Peipp

et al. 2014

The Journal of Immunology

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“…The chemical crosslinking is requested for stabilization of the samples for at least 2 weeks and shipping overseas for centralized flow cytometry analysis. For specific purposes (e.g., working with solid tumors, lymph nodes, tonsils, adherent cell lines, etc), the samples may need to be dilacerated or at least conveniently prepared before purpose (17,18). All steps, including incubation time and temperature need to be carefully controlled.…”

Section: Materials and Methods General Considerations For Receptor Ocmentioning

confidence: 99%

How validated receptor occupancy flow cytometry assays can impact decisions and support drug development

Moulard¹,

Ozoux

2015

Cytometry Part B Clinical

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Because of the pressure of significant attrition in drug development, demonstration of target engagement after drug administration enables dose and regimen optimization, patient selection, and stratification from the earliest stages of drug development. The determination of receptor occupancy (RO) can support these efforts. Flow cytometry is one of the preferred technologies to be used based on the important advances in the technology over the last years enabling the simultaneous determination on target cells, of multi intra or surface cell parameters with adequate precision in a regulated environment. Nevertheless, compared to other platforms using the same antigen-antibody binding concept, the flow cytometry approach has faced several challenges, not only due to the technology per se and the diversity of receptor occupancy approaches, but also related to the nature of the matrix where the determination is performed. To illustrate these points, three case studies (antibody-drug conjugate and naked antibody) are provided here to highlight the importance of the choice of the right antibody pair to measure both receptor density (RD) and occupancy by the drug on cancer cells in blood and in bone marrow and the possibility to circumvent the lack of a critical reagent with an innovative approach. In addition, the use of RO data to determine the minimum anticipated biological effect level (MABEL) with translational data from preclinical to human studies, selection of starting dose for the first in man study will be discussed. In the last couple of years, the advent of molecular medicine and targeted therapy has opened up innovative areas of investigation, allowing not only the development of new highly specific drugs but also a concomitant progress for the discovery of new approaches. These approaches fit perfectly with the development of antibody-based therapeutics since the drug and its target are clearly identified and unique. Nevertheless, the clinical development of targeted therapies (naked antibodies, immunoconjugates, etc.) remains disappointing since the average response rate has generally been low in human, at around 15% of patients (1). A better characterization of drug mechanism of action and target engagement coupled with a better understanding of the relationship between drug pharmacokinetic and pharmacodynamics is needed (2). Robust data to support dose selection and optimization of regimen before pivotal trials are critical to substantially shorten the time to reach strategic points during the clinical development. The use of RO in early phases of the drug discovery process to demonstrate proof of mechanism of the drug could eventually reduce the development risks, including the Cytometry Part B (Clinical Cytometry) 90B: 150-158 (2016)

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Fc Engineering

Peipp

Derer

Lohse

et al. 2014

Handbook of Therapeutic Antibodies

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Impact of Epidermal Growth Factor Receptor (EGFR) Cell Surface Expression Levels on Effector Mechanisms of EGFR Antibodies

Cited by 58 publications

References 51 publications

Epidermal Growth Factor Receptor Targeting IgG3 Triggers Complement-Mediated Lysis of Decay-Accelerating Factor Expressing Tumor Cells through the Alternative Pathway Amplification Loop

Epidermal Growth Factor Receptor Targeting IgG3 Triggers Complement-Mediated Lysis of Decay-Accelerating Factor Expressing Tumor Cells through the Alternative Pathway Amplification Loop

How validated receptor occupancy flow cytometry assays can impact decisions and support drug development

Fc Engineering

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