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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Published: January 19th, 2019

DOI:

10.3791/58291

1Centre de Recherche en Cancérologie et Immunologie Nantes Angers (CRCINA), Institut national de la santé et de la recherche médicale (INSERM), Université d'Angers, Université de Nantes, Nantes, France, 2Service de chirurgie pédiatrique, Centre hospitalier universitaire (CHU) de Nantes, Nantes, France, 3Unité de Formation et Recherche (UFR) des Sciences Pharmaceutiques et Biologiques, Université de Nantes, Nantes, France
* These authors contributed equally

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of <1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

The conventional approach in the treatment of a large number of different types of cancer was based on monotherapy. Even if it is still used in many cases, this method met several obstacles leading to opting for combined therapies2. Particularly, cancer cells are more susceptible to develop resistance when treated with a single drug by inducing alternative survival mechanisms3, resulting in therapeutic failure in patients4. Moreover, in monotherapy, drugs are usually administrated at a high dose. This situation often results in the occurrence of strong dose-dependent side effects that can be intol....

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Animal housing and experimental procedure were approved by the French Government (agreements #C44-278 and #APAFIS 03479.01). Animal care and procedures were conducted under directive EU 2010/63/EU and French Law #2013-118 on the protection of animals used for scientific purposes.

1. Evaluation of the Drug Interaction Between mAb 8B6 and Topotecan In Vitro

  1. 96-well sample preparation
    CAUTION: Consult the institution's Health and Safety committee a.......

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The representative results and figures are adapted with permission from earlier published work14.

Anti-OAcGD2 mAb 8B6 Synergistically Enhances the Inhibitory Effects of Topotecan on Neuroblastoma Cell Line Growth:

To establish the drug and the antibody concentrations to be used for assessing synergism between topotecan and mAb 8.......

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To predict the effect of drug interactions, three methods can be used: the isobologram methodology17, the nonlinear mixture model18, and the combination index1. Combination index analysis is the most commonly used because its application is simplified by the availability of a user-friendly computer program. For this purpose, we first characterized the dose-effect response of each agent used alone or in combination, by performing an MTT assay

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Grant support: Fondation de Projet de L'Université de Nantes, les Bagouz' à Manon, La Ligue contre le Cancer comité de Loire-Atlantique, comité du Morbihan, and comité de Vendée, une rose pour S.A.R.A.H, L'Etoile de Martin and la Société Française de Lutte contre les Cancers et les leucémies de L'Enfant et de L'adolescent (SFCE). M.B. and J.F. are supported by La Ligue Contre Le Cancer. The authors thank the UTE-facility of the Structure Fédérative de Recherche François Bonamy. The authors also thank Dr. S. Suzin (Inserm, Paris) for providing the IMR5 cells and Ms. H. Estéph....

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Name Company Catalog Number Comments
Cell Proliferation kit (MTT) Roche 11-465-007-001
CompuSyn software ComboSyn Combosyn can be downloaded for free at http://www.combosyn.com
Electric shaver Bioseb BIO-1556
Fetal calf serum Eurobio CVFSVFF00-01 10% heat-inactivated fetal calf serum in RPMI 1640
Firefox  Mozilla Corporation Firefox can be downloaded for free at http://www.mozilla.org/en-US/firefox/
Heat lamp Verre&Quartz 4003/1R
Human neuroblastoma IMR-5 cell line Accegen Biotechnology ABC-TC0450 IMR-5 is a clone of the human neuroblastoma cell line IMR32 5459762. IMR-5 cells were generously provided by Dr. Santos Susin (U.872, Paris, France)
L-glutamine Gibco 25030-024 2 mM in RPMI 1640
Lysis solution  Roche  11-465-007-001
mAb 8B6 University of Nantes N/A
Matrigel Corning 354248
Multiskan FC Thermofischer Scientific  N08625
Needle 21G 1 ½  BD Microlance 304432
Needle 25G 1 Terumo NN-2525R
NSG mice Charles River Laboratories 5557
Nunc MicroWell 96-well microplates Thermofisher 167008
PBS VWR L182-10
PBS, 0,05% EDTA Sigma-Aldrich E9884
PC that runs windows 7 Microsoft Windows 7 can be purchased at http://www.microsoft.com/en-gb/software-download/windows7
Penicillin-Streptomycin Gibco 15140-122 100 units/mL penicillin and 100 mg/mL streptomycin in RPMI 1640
Reagent reservoir Thermofischer Scientific 8094
Rodent restrainer Bioseb TV-150-SM
RPMI 1640 Gibco 31870-025
Syringe 1 mL Henke Sass Wolf 5010.200V0
Topotecan Sigma-Aldrich T2705

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