Immunoconjugate design: a predictive approach for coupling of daunomycin to monoclonal antibodies.

There is increasing interest in the development of daunomycin-antibody immunoconjugates for the targeting of drug to specific cells or tissues. To this end, we have examined the factors influencing the synthesis of daunomycin-monoclonal antibody conjugates linked covalently by an acid-labile cis-aconitic spacer (which is considered to aid drug release from immunoconjugates in the lysosomes and thus enhance their cytotoxic potential). A rapid and efficient procedure for the purification of drug from contaminants and stabilizers was first developed; conditions for the optimal preparation of cis-aconityldaunomycin were established; products were analyzed and identified by TLC and HPLC. The coupling of cis-aconityldaunomycin to antibody was accomplished by activating the modified drug with a carbodiimide before addition to antibody. Several factors were identified which influenced the efficiency of the conjugation; in particular, the compositional features of the antibody which determine its electrophoretic charge characteristics were of profound effect. However, by appropriate choice and control of buffer pH during conjugation, it was possible to define conditions resulting in the controlled substitution of antibody with drug. The consequent effects upon the cell-binding activity of immunoconjugates were established and related to the extent of substitution. The procedures described enable appropriate reaction conditions to be selected for the linkage of daunomycin to antibody (at set drug/antibody molar ratios) and in good yield, based upon consideration of the compositional and charge properties of the antibody.