Disruption of P‐glycoprotein anticancer drug efflux activity by a small recombinant single‐chain Fv antibody fragment targeted to an extracellular epitope

Abstract

Inherent and acquired MDR is characterized by simultaneous resistance to diverse anticancer drugs and continues to be a major impediment in the curative chemotherapy of cancer. The MDR1 gene product, Pgp, is an ATP-driven efflux pump, which extrudes a variety of dissimilar hydrophobic cytotoxic compounds from MDR cells. Pgp overexpression results in MDR of tumor cell lines in vitro as well as of a variety of human malignancies. Thus, one major goal is to develop strategies aimed at specifically disrupting Pgp drugefflux activity. To this end, we have developed a small recombinant antibody capable of potent reversal of MDR, by disrupting Pgp drug-efflux activity. Using a phage display approach, we isolated a small scFv recombinant antibody fragment that specifically reacts with the first extracellular loop of human Pgp. This scFv fragment binds specifically to various Pgp-overexpressing human MDR carcinoma cell lines, consequently disrupts Pgp drug-efflux function and thereby reverses the MDR phenotype. We have successfully disrupted anticancer drug-extrusion pump activity in MDR cells using a small recombinant scFv fragment. We propose that these novel small Fv-based recombinant antibody molecules may lead to the development of a new class of antibody fragment-based agents that specifically inhibit Pgp drug extrusion. Hence, these small recombinant antibody fragments may be applied in combination chemotherapy to overcome MDR in various human cancers. © 2004 Wiley-Liss, Inc. Key words: P-glycoprotein; multidrug resistance; single-chain FvCombination chemotherapy continues to play a major role in the treatment of various human malignancies. MDR, whereby tumor cells simultaneously possess intrinsic or acquired cross-resistance to diverse anticancer drugs, hampers the efficacy of the chemotherapeutic treatment of various human cancers. 1-3 Intense molecular investigations of the MDR phenomenon over the past 2 decades have resulted in the isolation and characterization of genes encoding for several plasma membrane glycoproteins associated with MDR. The best-studied mechanism of MDR in cancer cells is overexpression of an energy-dependent efflux pump, the multidrug transporter Pgp. 4 Pgp is a member of a large ABC superfamily of transport proteins. 5 Pgp-mediated MDR plays an important clinical role in the resistance of various tumor cells to chemotherapy, as judged by the high incidence of MDR1 expression in tumors of various types and the correlations between MDR1 expression and lack of response to chemotherapy in various human malignancies. 6,7 These findings prompted a major effort aimed at identifying strategies and agents capable of reversing Pgp-mediated MDR. 3 Inhibitors of the MDR phenotype in cancer cells may function in 2 main modes: they can either modify the expression (i.e., decrease it) or disrupt the drug-efflux function of the transporter proteins involved in MDR. 3 The search for "chemosensitizers" that disrupt the function of these drug exporters and thereby reverse MDR has grown in parallel with th...

Pluronics and MDR Reversal: An Update

“…7–9 Third, use of antibodies and antibody fragments to target and inhibit drug efflux transporters. 10–12 Fourth, silencing of the gene expression of the drug efflux transporters 13–15 or antiapoptotic proteins, such as BCL2 13,16 using antisense oligonucleotides, siRNA, or micro RNA. Fifth, use of small molecules to suppress non-ABC transporter-mediated resistance.…”

Antibodies and their Fragments as Anti-Cancer Agents

“…Due to these traits, over-expressed multidrug resistance proteins are excellent targets for antibody mediated cancer targeting. [49,50] (Table 1)…”

Recent Advances on the Molecular Mechanisms Involved in the Drug Resistance of Cancer Cells and Novel Targeting Therapies

“…Strategies developed to reverse MDR include the use of pharmacological agents that can interact with one or several ABC transporters, thereby inhibiting their functions, monoclonal antibody directed against a specific ABC transporter, and antisense oligonucleotide or oligodeoxynucleotide ( and Table 2 ). 17 , 24 , 103 , 112 , 113 , 114 , 115 , 116 , 117 , 118 An alternative approach also considered using agents that can inhibit the signaling cascade elements responsible for the enhanced expression of ABC transporters in resistant cancer cells 114 . Certain pharmacological agents have been identified and observed to inhibit the transport activity of P‐gp, MRPs, and BCRP/ABCG2 efflux pumps, increase the intracellular drug accumulation, and reverse MDR in vitro and in vivo , and in certain cases have reached clinical trials.…”