Targeting an extracellular epitope of the human multidrug resistance protein 1 (MRP1) in malignant cells with a novel recombinant single chain Fv antibody

Abstract

Inherent and acquired multidrug resistance (MDR) is characterized by a simultaneous resistance to diverse anticancer drugs and is a major impediment towards curative chemotherapy of cancer. Hence one important goal is to develop strategies aimed at specific targeting of major anticancer drug efflux transporters of the ATP-binding cassette (ABC) superfamily including multidrug resistance protein 1 -MRP1 (ABCC1). To date, no monoclonal antibody has been isolated that can target an extracellular MRP1 epitope. Using a phage display approach, we have isolated a recombinant singlechain Fv (scFv) antibody that specifically reacts with the extracellular N-terminus of the human MRP1. Flow cytometric analysis revealed that this scFv fragment binds specifically to various viable human tumor cells that display variable Key words: multi drug resistance (MDR); scFv antibody; MRP1; phage displayCombination chemotherapy continues to play a major role in the treatment of various human malignancies. However, the efficacy of various chemotherapeutics has been limited by the frequent emergence of various anticancer drug resistance phenomena. The best-studied mechanisms of multidrug resistance (MDR) in malignant cells involve the overexpression of ATP-driven anticancer drug efflux pumps of the ABC superfamily. 1-3 These include the multidrug transporter, P-glycoprotein (Pgp), which extrudes various hydrophobic cytotoxic agents, as well as members of the multidrug resistance protein (MRP) family, currently comprising 9 exporters (MRP1 through MRP9). 3-5 MRP1 was discovered during a differential hybridization screen aimed at identifying mRNAs overexpressed in doxorubicin-resistant lung cancer cells that lack P-glycoprotein overexpression. 6 The 100 -200-fold MRP1 mRNA overexpression was a result of gene amplification. Analysis of the 1,531 amino acid sequence of MRP1 sequence identified this protein as a member of the ATP-binding cassette (ABC) superfamily of transporter proteins. 6 Importantly, transfection of the MRP1 cDNA conferred upon sensitive cells MDR to anthracyclines, Vinca alkaloids, etoposide, arsenical and antimonial oxyanions but not to cisplatinum and mitoxantrone. 7 These studies paved the way for the isolation of additional MRPs, MRP2 through MRP9, which have been shown to confer resistance to various toxic compounds that are either initially hydrophilic or that undergo intracellular conjugation to glutathione, sulfate and glucuronate. 5 Furthermore, another ABC transporter recently shown to mediate MDR in various tumor cells of epithelial origin is the breast cancer resistance protein (BCRP). 8 In order to study anticancer drug transporter proteins such as MRP1 in clinical specimens and to reveal their physiological functions, specific monoclonal antibodies (MAbs) were recently developed. 9 -11 Monoclonal antibodies against MDR proteins were used for 2 main purposes: a) detection and quantification of MRP and P-glycoprotein expression in drug-resistant cancer cells and b) reversal of MDR in cancer cells by abolishin...

Membrane topology of human ABC proteins

“…The efforts of generating antibodies recognizing extracellular epitope(s) in ABCC1 and in other ABCC-like pumps were unsuccessful indicating that the extracellular loops and the extracellular amino-terminal segment of the ''long-ABCCs'' are relatively short and/or buried. However, recently, using a phage display approach, a recombinant single-chain Fv (scFv) antibody that specifically recognizes the extracellular N-terminal tail of the human ABCC1 has been developed [40].…”

The role of MRP1 in the multidrug resistance of colorectal cancer

“…The present study demonstrated the potential of MRP1 as a therapeutic target in the treatment of CRC with a MDR phenotype and will prompt efforts to develop agents that are able to repress MRP1-mediated drug transport. Currently, an antibody selective for MRP1 has been isolated; however, its specificity and efficiency as a chemosensitizer in the treatment of CRC has not yet been reported (17). Another potential treatment option is the post-transcriptional interference of MRP1 expression, as suggested by the results of the present study.…”

Probing ATP-dependent conformational changes in the multidrug resistance protein 1 (MRP1/ABCC1) in live tumor cells with a novel recombinant single-chain Fv antibody targeted to the extracellular N-terminus

“…Maxisorb immunoplates were coated with biotinylated peptides (10 μg/ml) that were captured on BSA‐Biotin (10 μg/ml; Sigma, St. Louis, MO), streptavidin‐coated wells (10 μg/ml; Promega, Madison, WI) by an overnight incubation at 4°C. We used the MRP1‐derived peptide 1: M 1 ALRGFCSADGSDPLWDW,18 which was specifically targeted by the recombinant A5scFv antibody, and several other MRP1 or Pgp‐derived peptides, as described previously 12. Following blocking with 2% skim‐milk in PBS (Biological Industries, Beth‐Haemek, Israel), the purified A5scFv‐PE38 immunotoxin (in sequential dilutions) was added for 1 hr at room temperature.…”

“…We further showed that one of these recombinant antibodies (termed A2scFV) abolished the drug efflux activity of Pgp and thereby reversed the MDR phenotype of human carcinoma cells to doxorubicin 11. Extending on these studies, we recently isolated another recombinant scFv antibody (termed A5scFV) that specifically targets an extracellular N‐terminal epitope of the human MRP1 as expressed on the surface of viable tumor cells with MRP1 overexpression 12. In an attempt specifically to target and eradicate MDR malignant cells with MRP1 overexpression, we fused the A5scFv antibody to Pseudomonas exotoxin.…”