Therapeutic potential of nimotuzumab PEGylated-maytansine antibody drug conjugates against EGFR positive xenograft

Abstract

// Siddesh V. Hartimath 1, 2 , Ayman El-Sayed 3 , Amal Makhlouf 1, 2, 4 , Wendy Bernhard 3 , Carolina Gonzalez 3 , Wayne Hill 3 , Angel Casaco Parada 5 , Kris Barreto 3 , Clarence Ronald Geyer 3 and Humphrey Fonge 1, 2, 6 1 Department of Medical Imaging, College of Medicine, University of Saskatchewan, Saskatoon SK, S7N 0W8, Canada 2 Saskatchewan Centre for Cyclotron Sciences (SCCS), the Fedoruk Centre, Saskatoon SK, S7N 5C4, Canada 3 Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon SK, S7N 5E5, Canada 4 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-Aini, 12411, Cairo, Egypt 5 Centre for Molecular Immunology, Havana, 11600, Cuba 6 Department of Medical Imaging, Royal University Hospital Saskatoon, Saskatoon SK, S7N 0W8, Canada Correspondence to: Humphrey Fonge, email: humphrey.fonge@usask.ca Clarence Ronald Geyer, email: ron.geyer@usask.ca Keywords: epidermal growth factor receptor I; antibody drug conjugates; near infrared imaging; PEGylated maytansine; colorectal cancer Received: September 07, 2018      Accepted: December 20, 2018      Published: February 01, 2019 ABSTRACT Nimotuzumab is a humanized anti-epidermal growth factor receptor I (EGFR) monoclonal antibody. We have developed antibody drug conjugates (ADCs) with nimotuzumab conjugated to PEGylated-maytansine (PEG 6 -DM1). We generated conjugates with low (nimotuzumab-PEG 6 -DM1-Low: DAR = 3.5) and high (nimotuzumab-PEG 6 -DM1-High: DAR = 7.3) drug to antibody ratios (DAR). Quality control was performed using UV spectrophotometry, size exclusion HPLC, bioanalyzer, biolayer interferometry (BLI), and flow cytometry in EGFR-positive DLD-1, MDA-MB-468 (high density EGFR), and HT-29 (very low EGFR density) cells. Control antibody drug conjugates were developed using a human anti-maltose binding protein (MBP) antibody. BLI showed that the binding of nimotuzumab-PEG 6 -DM1-Low and nimotuzumab-PEG 6 -DM1-High was slightly but significantly affected by conjugation of the drug (nimotuzumab K D 0.89 ± 0.02 nM < nimotuzumab-PEG 6 -DM1-Low K D 1.94 ± 0.02 nM < nimotuzumab-PEG 6 -DM1-High K D 3.75 ± 0.03 nM). In vitro cytotoxicity was determined following incubation of cells with the immunoconjugates and IC 50 values were determined. Nimotuzumab-PEG 6 -DM1-Low and nimotuzumab-PEG 6 -DM1-High were used to treat EGFR positive KRAS mutant DLD-1 colorectal cancer xenograft. DLD-1 cells were transduced with a red fluorescent protein (iRFP702) to allow the use of near infrared imaging (NIR) for tumor response monitoring. In vitro potency correlated with the number of drugs on antibody, with nimotuzumab-PEG 6 -DM1-High showing higher activity than nimotuzumab-PEG 6 -DM1-Low. Three doses (15 mg/kg) of the ADCs prolonged the survival of DLD-1-iRFP-702 tumor bearing mice as monitored by NIR. Nimotuzumab-PEG 6 -DM1-Low resulted in 4/6 complete cure while nimotuzumab-PEG 6 -DM1-High resulted in 2/5 complete cure. The novel ADCs were very effective in a colorectal cancer model in vivo .