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

Abstract

In an attempt to design cancer chemotherapeutic agents of improved specificity by linking them to antibodies against tumor-associated antigens, we 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 ECDI and active ester methods yielded covalent conjugates as established by: ( a ) lack of binding of MTX to IgG in the absence of ECDI or when MTX was added without prior activation; and ( b ) the presence of a constant amount of MTX in the IgG fraction after gel filtration. The active ester method was the most effective. Ten mol of MTX per mol of IgG could be incorporated with retention of 90% of the anti-bovine serum albumin activity of an equimolar amount of unconjugated IgG and with recovery of 70% of the original protein. In the ECDI procedure, there was 50% loss of protein, and the recovered protein retained only 50% antibody activity at an incorporation level of 8 mol of MTX per mol of IgG. Conjugates containing 12 mol of drug per mol of IgG lost all antibody activity. MTX conjugated by both the active ester and ECDI methods partially retained the ability to inhibit dihydrofolate reductase. The mixed anhydride procedure failed to incorporate more than 2 to 3 mol of MTX per mol of IgG even when there was a 200-fold molar excess of anhydride in the reaction mixture. The conjugate retained full antibody activity, but neither it nor the product resulting from the reaction of MTX with acetic anhydride inhibited dihydrofolate reductase. 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. Conjugates containing 13 mol of MTX per mol of IgG retained their reactivity with EL4 cells on membrane immunofluorescence assay. When tumor-inoculated mice (104 EL4 cells/animal) were given injections of this conjugate on Days 1, 4, and 7 after inoculation (MTX dose, 4 mg/kg/injection), they survived significantly longer than did mice treated with equivalent amounts of drug alone, antitumor IgG alone, drug followed by antibody, or drug linked to non-tumor-specific IgG. 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 ± 2.47 (S.D.) days after tumor inoculation.