Covalent binding of methotrexate to immunoglobulins and the effect of antibody-linked drug on tumor growth in vivo

In an attempt to design cancer chemotherapeutic agents of improved specificity by linking them to antibodies against tumor-associated antigens, the authors studied the binding of methotrexate (MTX) to immunoglobulins by three different methods, initially using a model system that consisted of bovine serum albumin and rabbit anti-bovine serum albumin immunoglobulin G (IgG). One method used coupling via water-soluble 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (ECDI), whereas two others used reactive intermediate derivatives of MTX, i.e., an active ester formed by reaction with N-hydroxysuccinimide or a mixed anhydride formed by reaction with acetic anhydride. The demonstrated superiority of the active ester method in producing active conjugates prompted us to use this technique for linking MTX to a rabbit IgG antibody against the mouse EL4 lymphoma. Eight of 11 mice treated with this MTX-anti-EL4 IgG conjugate survived tumor free for an observation period of 90 days, whereas control mice died at 17.3 plus or minus 2.47 (S.D.) days after tumor inoculation.