The generation of recombinant phage antibodies to the multiple drug resistance protein P-glycoprotein
The generation of human antibodies to the multiple drug resistance protein, P-glycoprotein (Pgp), has been a challenge in the field of cancer chemotherapy since drug resistance was first described. Pgp was identified as being an intrinsic factor in the resistance to chemotherapeutic drugs, as its levels were found to escalate after primary drug challenges. Further investigation into the structure and function of P-glycoprotein elucidated that its activity was ATP dependent, and that it had broad spectrum specificity. In order to augment the success of chemotherapeutic treatment of patients it was deemed necessary to regulate the activity of this protein. This was possible by physical inhibition of its activity or through regulation at the genetic level. This project addresses the former of the two options, the physical inhibition of Pgp activity. It was considered that by the generation of antibodies to the extracellular regions of Pgp it would be possible to inhibit its activities. For such antibodies to be effective in the human model, the antibodies would have to bear human determinants in order that the human immune system would not attach and destroy these antibodies before they had the desired effect. Therefore it was decided that recombinant phage technology should be used. This is a system which selects single chain antibodies with human determinants from a large and diverse population of antibodies with a variety of specificities. Initially, attempts were made to generate a phage library de novo using a commercial kit designed for this purpose. However, after repeated attempts at this process, this approach was found to be beyond technical abilities, and was rejected in favour of a more rudimentary approach. Two libraries were screened for antibodies which would bind to membrane preparations from two leukaemic cell lines, CCRF CEM and CCRF CEM VLB. The former cell line was a drug-sensitive cell line which responded favourably to drug challenge, and the latter was a drug-resistant cell line which thrived in drug-rich environments. The drug-resistant cell line was derived from the drug-sensitive cell line, and was considered to be identical in all respects except drug sensitivity. Antibodies obtained from a large multicombinatorial library, the Lox library, were found to display selective binding to CCRF CEM VLB cell membranes in favour of CCRF CEM cell membranes. Antibodies were also generated to peptides representing the 6 extracellular loops of Pgp. From these selections antibodies were generated which were found to selectively bind the peptide to which they had been raised.