A new approach to bifunctional chelate attachment to antibodies

One potential problem with the synthesis of bifunctional chelates (BC) of antibodies (Abs) is inactivation of the Ab by attachment of the chelate (C) at or near the antigen (Ag) binding site. The most common method of synthesizing BC depends on attachment of a C (e.g., DTPA) to a free amino group on the Ab molecule. However, the Ab may be inactivated if this amino group is too near the Ag binding site. We examined an alternative method, with attachment of a C, desferrioxamine-B(DF), to free carboxyl groups of Ab molecules. BC of a monoclonal Ab to a melanoma-associated Ag were prepared using DF, and labeled with In-111. DF-Ab was prepared by mixing Ab, DF, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at pH 4.75. BC of the same Ab also were prepared, using DTPA, by the cyclic anhydride method (DTPA-Ab). Good radiolabeling yields were achieved with both DF-Ab and DTPA-Ab. The reactivity of DF-Ab and DTPA-Ab with melanoma Ag was treated in vitro. Binding of the Ab to melanoma cells and to control (lymphoma) cells was assayed. DF-Ab and DTPA-Ab demonstrated significant cell binding (61.5 and 38.2% respectively, at appropriate dilutions) when tested with melanoma cells. Neither Ab bound significantly to control cells (6.9 and 3.3% respectively). These experiments demonstrate that BC of Abs can be successfully prepared by binding C to free carboxyl groups on Abs. The DF-Ab so produced demonstrates significant reactivity with its Ag. With some Abs, as with the above anti-melanoma Ab, this method of BC preparation may result in less loss of antigenic reactivity than occurs with conventional methods