Site‐specific linkage of methotrexate to monoclonal antibodies using an intermediate carrier

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Abstract
The site‐specific conjugation of methotrexate, 4‐amino‐N10‐methylpteroylglutamic acid, to a monoclonal anti‐carcinoembryonic antigen (CEA) antibody, using an intermediate aminodextran carrier system, resulted in a ratio of 30‐50 molecules of MTX per molecule of immunoglobulin. The immunoreactivity of the conjugate was analyzed using flow cytometry or a competitive binding assay, which showed that the conjugate has significant retention of the antigen‐binding activity. The pharmacokinetic behavior of the immunoconjugate in BALB/c mice and tumor localization in hamsters indicated that the conjugate remained in the circulation with higher concentration than free antibody, and could recognize the tumor as efficiently as the unconjugated antibody. The high degree of drug incorporation with retained immunoreactivity makes this method preferable to direct antibody conjugation.