Internal Image-Bearing Anti-ldiotypic Monoclonal Antibodies

Abstract

Five anti-idiotypic (Id) monoclonal antibodies (MAbs) (Ab2) were prepared from a BALB/c mouse immunized with anti-carcinoembryonic antigen (CEA) MAb MA208 (Ab1) in a syngeneic system. These anti-Id MAbs appear to recognize unique idiotopes at the combining site of MAb MA208, because they were specifically reactive with MAb MA208 and showed inhibitory activity against the binding of MAb MA208 to CEA. These MAbs were divided into three groups according to the analysis of anti-anti-Id antibodies (Ab3) induced with each anti-Id MAb. Anti-anti-Id MAb M7-625 antiserum (Ab3) reacted with purified CEA in a binding assay and in Western blot analysis, and competed with Ab1 binding to CEA. Furthermore, the binding of anti-Id MAb M7-625 to MAb MA208 was inhibited with CEA, indicating that Ab2 mimics the structure of the epitope in CEA which was recognized with Ab1. These serologic findings suggest that anti-Id MAb M7-625 carries the internal image of the antigen. According to the amino acid sequences of complementarity determining region (CDR) 1, 2 and 3 of the MAb M7-625 variable region, homology of amino acid sequences exists between CDR2 in the H chain (5 amino acids of 10) and domain III of CEA (545-554). Seven anti-Id MAbs were then generated using anti-CEA synthetic peptide MAb P1-356 to analyze further the epitope structure of CEA. These anti-Id MAbs were divided into four groups. Serological analyses as described above suggested that among them, anti-Id MAb M315 had an internal image. We therefore prepared anti-anti-Id MAbs using anti-Id MAb M315. Among them, anti-anti-Id MAb 11B2 reacted directly with CEA and competed with MAb P1-356 in the competition assay. In addition, MAb 11B2 stained both cultured CEA-producing cells and colonic cancer tissues, suggesting that MAb 11B2 is Ab1 like Ab3. These MAbs (Ab1-3) will be of use for the structural analysis of the internal image.