| The long term goal of this project is to develop a method for breast cancer treatment that uses tumor-specific libraries of recombinant polyclonal antibodies. The selection of polyclonal antibody libraries with desired specificities is made possible by the technology for generating Fab phage display libraries. Variable (V) region genes for generating Fab phage display libraries were obtained from mice (a conventional source) or chickens (a non-conventional source).; A model system was developed in which chickens were immunized with sheep red blood cells (SRBCs) and a diverse population of V region genes was amplified by PCR, using a single set of primers. After insertion of the chicken V region genes into a phage display vector, chimeric Fab with chicken V regions and mouse constant (C) regions was expressed on the phage surface. The chimeric phage were positively selected for binding to SRBCs, and the VL-VH region gene pairs of the selected library were transferred in bulk to a mammalian expression vector. Chimeric whole antibodies with chicken V regions and mouse C regions were expressed.; One potential problem with the phage system is the outgrowth of clones which do not express Fab on their surface. This was circumvented through the addition of a gene encoding the Zeocin resistance selectable marker into the leader-promoter cassette. Because of the vector construction, the leader-promoter cassette is the last DNA segment to be incorporated into the phage display vector. With the addition of the antibiotic Zeocin, only those phage expressing the entire construct for Fab production survive. This was demonstrated in a model system of phage specific for the hapten p-azophenylarsonate.; Finally, the human breast carcinoma cell line BT-20 was used for production of polyclonal antibody libraries against breast carcinoma cells. After immunization of mice and chickens with BT-20 cells, diverse populations of V region genes were amplified, as confirmed through nucleotide sequence analysis. Fab-phage display libraries ({dollar}>{dollar}10{dollar}sp7{dollar} members) were constructed and selected on BT-20 cells, and specificity for breast carcinoma cells was demonstrated by ELISA and inhibition ELISA. The selected VL-VH region gene pairs are available to be transferred in bulk to a mammalian expression vector for expression as whole anti-tumor polyclonal antibodies. |
