Yumiko Takeda

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.

Target-selective cytotoxicity of methotrexate conjugated with monoclonal anti-MM46 antibody.

SummaryIn studies on antitumor antibody-cytotoxic drug conjugates as potential tumor-selective cytotoxic agents, methotrexate (MTX) was conjugated via its active ester derivative with a murine monoclonal antibody (aMM46) to a mouse mammary tumor antigen (MM antigen) on syngeneic, ascitic C3H/He mouse mammary tumor MM46 cells. The conjugate retained full antibody activity, as assayed by complement-dependent cytolysis. The target-selective cytotoxicity of aMM46-MTX was verified by the observations that this conjugate showed greater cytotoxicity than the corresponding normal mouse immunoglobulin (nIg) conjugate to MM46 cells, neither aMM46 nor nIg being cytotoxic, and that it showed less cytotoxicity to MM antigen negative mouse mammary tumor MM48 cells than to MM46 cells, its cytotoxicity to MM48 cells being similar to that of the nIg conjugate. From the results of assays of cell binding and uptake of 131I-labeled aMM46 and aMM46-3H-MTX, aMM46 and aMM46-MTX were internalized after their binding to MM46 cell surface antigen. Leupeptin, an inhibitor of the lysosomal endopeptidase cathepsin, decreased the cytotoxicity of aMM46-MTX, supporting the involvement of lysosomal degradation of the conjugate in its action.

Drug/Antibody Conjugates. In Vitro Cytotoxicity of a Human Serum Albumin‐Mediated Conjugate of Methotrexate with Anti‐MM46 Monoclonal Antibody

In studies on antitumor antibody-drug conjugates as potential antitumor agents with improved tumor-selectivity [ 1, 21 , we prepared a conjugate of methotrexate (MTX) with a monoclonal antibody (aMM46) to an antigen on ascitic C3H/He mouse mammary tumor MM46 cells (MM antigen) with human serum albumin (HSA) as an intermediary [3 ] . As shown in Fig. 1, using an active ester of MTX, we first linked MTX to HSA which had been conditioned to have about 1 mol of thiol group per mol of HSA. The resulting HSA-MTX was then reacted with aMM46 with the maleimide group introduced (MTX/HSA/IgG molar binding ratio, 28.0/ 1.16/ 1 .O). We examined the in vitro cytotoxicity of the conjugate aMM46-HSA-MTX by culturing MM46 cells or MM antigennegative MM48 cells, another C3H/He mouse mammary tumor cell line, with the conjugate and controls for two to three days followed by enumeration of the viable cells. MTX conjugated with