BIOTIN REAGENTS FOR ANTIBODY PRETARGETING .2. SYNTHESIS AND IN-VITRO EVALUATION OF BIOTIN DIMERS AND TRIMERS FOR CROSS-LINKING OF STREPTAVIDIN

Polymerization and/or cross-linking of recombinant streptavidin (r-SAv ) with biotin derivatives containing two biotin moieties (biotin dimer s) or three biotin moieties (biotin trimers) has been investigated as a model for reagents to be used to increase the amount of radioactivit y on cancer cells in tumor pretargeting protocols. In the investigatio n, six biotin dimers and three biotin trimers were synthesized. Most b iotin derivatives synthesized had ether containing Linker molecules in corporated to improve their aqueous solubility. The synthesized biotin dimers contained linker moieties which provided distances (when fully extended) of 13-49 Angstrom between biotin carboxylate carbon atoms, and the biotin trimers contained linker moieties which provided distan ces of 31-53 Angstrom between any two biotin carboxylate atoms. All of the biotin derivatives were evaluated for their ability to polymerize r-SAv in solution. When the biotin derivatives were mixed with r-SAv, none of the biotin dimers caused polymerization, but all of the bioti n trimers resulted in complete polymerization. Some of the biotin dime rs did cross-link r-SAv (to form r-SAv dimers, trimers, etc.), but the percentage of crosslinking was low (less than or equal to 40%). The l ength of the linker molecule was important in cross-linking of biotin dimers. While linkers which provided distances of 13 and 19 Angstrom b etween biotin carboxylate carbon atoms did not result in cross-linking , a linker which provided a 17 Angstrom distance resulted in a small ( less than or equal to 10%) amount of cross-linking. Also, cross-linkin g was increased in biotin dimers with linkers which provided distances between biotin carboxylate carbon atoms of greater than or equal to 2 3 Angstrom. Cross-linking of streptavidin bound in polystyrene wells w ith biotin dimers and trimers was also examined. In those experiments, an excess of each biotin derivative was incubated at 37 degrees C for 10-30 min in polystyrene wells containing bound SAv. After the excess biotin derivative was rinsed from the wells, an excess of r-[I-125]SA v was incubated for another 10-30 min. The amount of r-[I-125]SAv boun d after rinsing the excess from the wells was an indicator of the exte nt of cross-linking that occurred. The process of alternating addition s of reagents was repeated four times to demonstrate that bound radioa ctivity could be increased with each addition of [I-125]SAv. The resul ts of cross-linking r-SAv in polystyrene wells paralleled results from cross-linking in solution.