A quantitative flow cytometric assay for determining binding characteristics of chimeric, humanized and human antibodies in whole blood: Proof of principle with rituximab and ofatumumab

Engelberts, Patrick J.; Badoil, Carole; Beurskens, Frank J.; Boulay-Moine, Danièle; Grivel, Karine; Parren, Paul W.H.I.; Moulard, Maxime

doi:10.1016/j.jim.2012.11.007

Cited by 9 publications

(10 citation statements)

References 35 publications

(41 reference statements)

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“…Malignant cells frequently have altered cell-surface protein expression compared with normal cells ( 9 – 11 ). These expression differences have broad implications for target selection, tissue penetration, drug specificity, and efficacy, and in a clinical setting they have broad implications for the effects of diagnosis, disease monitoring, and treatment modulation ( 1 , 11 – 13 ). A number of recent reviews highlight the potential importance of intratumoral heterogeneity, likely intrinsic to many cancer types, as a source of resistance to currently available therapies ( 14 – 16 ).…”

Section: Introductionmentioning

confidence: 99%

“…We determined the cell-surface density of EGFR and c-MET in a panel of cancer cell lines grown under uniform conditions using flow cytometry. The application of flow cytometry methods to the quantitation of cell-surface antigens has become widespread since its introduction in the 1980s ( 13 , 25 ). The term quantitative flow cytometry (QFCM) was coined to describe a set of methodologies for quantitation designed to standardize procedures and reagents to minimize inter-laboratory variability ( 11 ).…”

Section: Introductionmentioning

confidence: 99%

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Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody

Jarantow

Bushey

Pardinas

et al. 2015

Journal of Biological Chemistry

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Background: Cancer cells express surface antigens at different levels from normal cells.Results: Differences in EGFR and c-MET receptor density levels influenced the in vitro activity of an EGFR × c-MET bispecific antibody.Conclusion: Consideration of target expression levels is important for bispecific design.Significance: In addition to multiple pathway targeting, the unique avidity of bispecific antibodies contributes to their promise for cancer therapy.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody

Jarantow

Bushey

Pardinas

et al. 2015

Journal of Biological Chemistry

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“…In the absence of a reliable noncompeting antibody (MAb2) to determine the total number of receptors in the presence of drug, a new approach was developed and validated to quantify both free and total receptors. This concept has been also validated for CD20 (23). The free CD19 density (MAb1 free ) was assessed with MAb1 (muaB4 clone).…”

Section: Cd19 Targeting Drug: Sar3419mentioning

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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“…This "quantitative FCM" (QFCM) approach, allowing absolute quantification of membrane antigens, has already found several experimental or clinical applications 19 . With the rise of biotherapies, such quantitative approach could be helpful in biopharmaceutical development [20][21][22] both to measure the expression of target antigens and to characterize new entities 23,24 . The aims of this work were first to develop an original QFCM assay to measure the number of antibodies coated on the surface of submicron-sized particles and second to evaluate the potential impact of antibodies coating level on immunoliposomes cytotoxic effect.…”

mentioning

confidence: 99%

Prototyping Trastuzumab Docetaxel Immunoliposomes with a New FCM-Based Method to Quantify Optimal Antibody Density on Nanoparticles

Rodallec¹,

Franco²,

Robert³

et al. 2020

Sci Rep

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Developing targeted nanoparticles is a rising strategy to improve drug delivery in oncology. Antibodies are the most commonly used targeting agents. However, determination of their optimal number at the surface remains a challenging issue, mainly due to the difficulties in measuring precisely surface coating levels when prototyping nanoparticles. We developed an original quantitative assay to measure the exact number of coated antibodies per nanoparticle. Using flow cytometry optimized for submicron particle analysis and beads covered with known amounts of human igG-kappa mimicking various amounts of antibodies, this new method was tested as part of the prototyping of docetaxel liposomes coated with trastuzumab against Her2+ breast cancer. This quantification method allowed to discriminate various batches of immunoliposomes depending on their trastuzumab density on nanoparticle surface (i.e., 330 (Immunoliposome-1), 480 (Immunoliposome-2) and 690 (Immunoliposome-3), p = 0.004, One-way ANOVA). Here we showed that optimal number of grafted antibodies on nanoparticles should be finely tuned and highest density of targeting agent is not necessarily associated with highest efficacy. Overall, this new method should help to better prototype third generation nanoparticles. Development of nanoparticles (NP) for targeted delivery and controlled drug release may improve the therapeutic index of drugs, especially that of anticancer agents. Such improvement is particularly relevant when administering cytotoxics that show frequently dose-limiting toxicities, thus resulting in suboptimal efficacy 1. In addition to passive targeting through the Enhanced Permeation and Retention effect 2,3 , active targeting is based upon receptor-mediated binding of nanoparticles to the membrane of tumor cells. A successful and actively targeted nanomedicine requires therefore a fine balance of ligand content and surface exposure of cell-binding moieties that minimize immunological recognition and rapid clearance by macrophages and scavenging cells. The current methods for post-synthesis NP surface modification often require multi-steps complicated procedures, thus making difficult to achieve batch-to-batch reproducibility 4-6. This calls for the need to develop post-synthesis analytical methods to control the surface properties of the NP, such as the exact coating rate when a monoclonal antibody is to be used as a targeting agent. To date, no such method has been made available and little information is usually provided regarding the exact number of monoclonal antibodies coated on third generation nanoparticles such as immunoliposomes. Assaying monoclonal antibodies in pharmaceutical preparations is mostly based upon Enzyme Linked ImmunoSorbent Assay (ELISA) techniques. Indirect methods such as Bradford assay or Pierce BCA protein assay have been proposed, but they can only provide semi-quantitative information and are in no way suitable to precisely measure the exact amount of antibodies grafted on a nanoparticle surface 7-10. Flow

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A quantitative flow cytometric assay for determining binding characteristics of chimeric, humanized and human antibodies in whole blood: Proof of principle with rituximab and ofatumumab

Cited by 9 publications

References 35 publications

Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody

Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody

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

Prototyping Trastuzumab Docetaxel Immunoliposomes with a New FCM-Based Method to Quantify Optimal Antibody Density on Nanoparticles

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