טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLiat Binyamin
SubjectTargeting Human Multidrug Resistance Protein 1(MRP1) with
Recombinant Antibodies
DepartmentDepartment of Biology
Supervisor Full Professor Reiter Yoram


Abstract

Combination chemotherapy continues to play a major role in the treatment of various malignancies. Human multi drug resistance protein 1 (MRP1) belongs to the ATP-binding cassette superfamily of transport proteins and is involved in conferring multidrug resistance of cancer cells to multiple chemotherapeutic agents. To facilitate both experimental and clinical studies of MRP1, several laboratories have generated MRP1-reactive-MAbs, however, to date, no mAb detecting external epitopes of the MRP1 have been reported. Our major goal was to develop antibody-based small molecules that can be used as extracellular probes for studying and targeting MRP1 on intact tumor cells.

Using a phage display approach, we have isolated a small single-chain Fv that specifically reacts with the extracellular N-terminus of the MRP1 on viable MRP1-expressing tumor cells. To explore the potential targeting capabilities of this scFv, it was used to construct an immunotoxin. The immunotoxin bound to immobilized MRP1-derived peptide but not to MRP1-expressiong cells. Thus, we concluded that the A5scFv has favorable binding capabilities to the referred epitope. Subsequently, the A5scFv was used as a molecular probe for detecting conformational changes that might occur during the catalytic cycle of transport. Results showed that the A5scFv stained cells in an ATP-dependent manner, indicating that the binding is influenced by conformational changes of the N-terminus of MRP1, which are presumably part of the transport mechanism. Our scFv is the first extracellular probe that can be further considered as a tool for understanding MRP1 transport mechanism leading to efficient modulators searching.