Characterization of Human Colorectal Cancer MDR1/P-gp Fab Antibody

Xuemei Zhang,Gary Guishan Xiao,Ying Gao

doi:10.1155/2013/716289

Xuemei Zhang, Gary Guishan Xiao + Show 1 more

Open Access

https://doi.org/10.1155/2013/716289

Copy DOI

Abstract

In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29). Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl β-D-1-thiogalactopyranoside (IPTG). After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT) comprising residues 883–898 within the transmembrane (TM) domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied.

Highlights

Failure of chemotherapy resulting from a multidrug resistance (MDR) remains the major obstacle during cancer treatment
Comparative sequence analysis of P-gp with other ABC family members shows that it consists of two transmembrane (TM) domains, each containing six putative TM segments followed by an ATP-binding consensus motif and two cytoplasmic nucleotide-binding domains (NBDs)
The peptide sized 21 kDa covering the P-gp transmembrane region was first prepared by us. Both anti-human colorectal cancer P-gp21 Fab antibody and its gene sequence have been successfully obtained by phage display technology

Summary

Introduction

Failure of chemotherapy resulting from a multidrug resistance (MDR) remains the major obstacle during cancer treatment. P-gp is a 170 kD plasma membrane efflux pump protein and belongs to the ATP-binding cassette superfamily of transport proteins [1]. Structure-function analyses of P-gp suggested that two subunits of P-glycoprotein are involved in the selection of its drug substrate and/or specificity of MDR modulator. P-gp functions as an efflux pump expelling drugs out of tumor cells, resulting in a decrement intracellular concentration of cytotoxic drugs. Use of the intact monoclonal antibodies may provide false positive signals in detection of the tumor cells since the Fc fragments of IgG may bind to the Fc receptors on the surface of normal cells as well. Fab is smaller than IgG in size, facilitating trafficking of the antigen across cell membrane and may provide more information than the intact antibody during the ICHA process [11, 12]

Methods

Discussion

Conclusion