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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 and follow local regulation rules related to laboratory safety. Review the Material and Safety Data Sheet information before working with any media, ce....

Results

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.......

Discussion

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

Materials

List of materials used in this article

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