Antibody phage libraries for the next generation of tumor targeting radioimmunotherapeutics

Pretargeting techniques have shown promise for enhancement of the therapeut ic index of radioimmunotherapy for cancer. However, methods to vary and com pare antibody configurations and select optimal combinations have proved ra ther formidable. New options for the construction of pretargeting molecules are provided by sophisticated use of the diversity and malleability of ant ibody genes. Diverse arrays of single-chain antibody fragments (scFvs) can now be obtained reactive with virtually any target antigen by selection fro m human naive phage antibody libraries. ScFvs can also be cloned directly f rom hybridoma for construction of phage libraries that facilitate subsequen t manipulation: e.g., affinity maturation and modification of specificity. ScFvs affinity selected from these sources to their specific antigen target s have demonstrated a wide spectrum of binding characteristics. ScFvs selec ted from a large human naive phage antibody library by binding Cu-1,4,8,11- tetracyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA) or Y-1,4,7,10-tet ra-azacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) have shown diversit y by DNA fingerprints. DNA sequence information confirmed that the anti-TET A scFv represented diverse scFv gene families, ScFvs for Y-DOTA and those f or lymphoma-associated HLA DR10 (Lym-1) were selected in a similar manner f rom mouse antibody gene libraries derived from hybridoma, ScFv clones for e ach of these antigens were chosen for further study based on the results of ELISA assays involving the respective cell membrane or metal chelate antig ens. A PCR primer system built to pCANTAB 5E expression vector sequence nas designed to facilitate cloning of antibody heavy (V-H) and light (V-L) gen es from selected scFvs as cassettes into diabody modules. Thus, chosen scFv s could be expressed in the same diabody format for comparative study. Sele cted mouse anti-DOTA scFv and Lym-1 scFv genes were linked as V-HA anti-DOT A-link-V-LB Lym-1; V-HB anti-DOTA-link-V-LA Lym-1 and ligated into the pCAN TAB 5E vector. Corresponding diabodies were expressed in Escherichia coli a nd purified by affinity chromatography. Here we provide a perspective on th e power of antibody phage libraries and the possibilities of creating simpl e molecular formats that can be used en route to the development of new tum or targeting and pretargeting molecules.