Development of a hyperimmune Anti-MUC-1 single chain antibody fragments phage display library for targeting breast cancer

Radioimmunotherapy (RIT) has demonstrated potential for improving clinical cancer therapy. Optimizing the approach has proven difficult thus far. Anti body phage display libraries provide unique molecules that could improve RI T, A phage display library of single chain antibody fragments (scFv) agains t the MUC-1 mucin molecule, which is expressed on 90% of human breast cance rs, was produced from the spleen cells of MUC-1 hyperimmunized BALB/c mice, Increased serum IgG levels, 15 times baseline, were detected following the third immunization. RNA from the spleen cells was isolated, cDNA was made, and variable heavy and variable light immunoglobulin chain gene regions we re amplified using PCR technology. The variable heavy and variable light ch ain gene regions were combined with a flexible linker, ligated into the pCA NTAB 5E phagemid vector, and electroporated into TG1 Escherichia coli cells . A library of 10(7) initial colonies was compiled. Forty-six of 288 coloni es screened for reactivity demonstrated binding to MUC-1-expressing MCF-7 b reast cancer cell membrane fragments. Anti-MUC-1 library diversity evaluate d by BstNI digest demonstrated that 52% of the anti-MUC-1 scFv binding MCF- 7 possessed individual banding patterns representative of approximately 5 x 10(5) colonies likely able to recognize distinct epitopes present on MUC-1 positive human breast cancers. In summary, the anti-MUC-1 scFv antibody ph age library contains diverse scFv molecules, which should provide unique ch aracteristics and epitope recognition. These molecules will be used in the development of pretargeting RIT strategies designed to improve the clinical outcome of patients with breast cancer.