Preparation and Characterization of Immunoconjugates for Antibody-Targeted Photolysis

Monoclonal antibody (MAb)-dextran-tin(IV) chlorin e6 (SnCe6) immunoconjugates were prepared by a new technique involving the use of reducing, terminal-modified dextran carriers and site-specific modification of the Fe oligosaccharide moiety on the antibodies. Dextran carriers were synthesized to increase the number of SnCe6 molecules attached to a MAb. The dextran carriers were coupled to the MAb via a single, chain-terminal hydrazide group to prevent aggregation of MAbs. Conjugates were prepared with antimelanoma MAb 2.1 containing up to 18.9 SnCe6 molecules per MAb. Under neutral conditions, no hydrolysis of the hydrazone bond between the MAb and the dextran carrier could be detected, and the hydrazone was not stabilized by reduction with NaCNBH3 or NaBH4. Analysis of the purified immunoconjugates showed that approximately two dextran carrier chains were attached to a MAb regardless of the number of SnCe6 molecules linked to a dextran carrier. Site-specific covalent attachment of the SnCe6-dextran chains to the MAb was confirmed by SDS-PAGE. HPLC analysis of the conjugates gave a single species eluting in the range of 200-240 kDa. As determined by a competitive inhibition radioimmunoassay using viable SK-MEL-2 human malignant melanoma cells, the conjugates showed excellent retention of antigen-binding activity relative to unconjugated MAb.