A novel covalent modification of antibodies at their amino groups with retention of antigen-binding activity

A novel method of covalent modification of antibodies at their amino groups with retention of antigen-binding activity is described. The procedure is as follows: (a) blockade of those amino groups of antibodies whose integrity is essential to their antigen-binding activity with 2,3-dimethylmaleic anhydride, a reversible amino group-blocking reagent; (b) modification of residual amino groups with reagents reactive with the amino groups; and (c) removal of dimethylmaleyl groups by hydrolysis. This procedure was used for covalent conjugation of methotrexate (MTX) with two monoclonal antibodies against human melanoma-associated antigens using MTX N-succinimidyl ester. MTX attached to the antibodies at sites other than the amino groups via less stable bond(s) was removed by treatment with hydroxylamine.