Comparison of multiple anti-CEA immunotoxins active against human adenocarcinoma cells

Abstract

Anti-carcinoembryonic antigen (CEA) immunotoxins constructed with multiple anti-CEA antibodies (goat and baboon polyclonal, and three murine monoclonal antibodies) by covalently linking them to the A chain of ricin via a disulfide bond all function as potent and specific toxins for CEA-bearing cells, suggesting that the CEA molecule is capable of directing productive internalization of ricin A chain. The high potency of anti-CEA immunotoxins apparently makes addition of ricin B chain unnecessary for high toxic efficiency, as in some other systems, because presence of the B chain reduces target cell specificity. Several characteristics of the immunotoxins which might account for their cytotoxic potency were studied. Equilibrium association constants of the goat, baboon, and murine monoclonal C-19 antibodies with fluid-phase CEA were determined by using Langmuir plots and were found to be 8.79, 6.61, and 8.13×10⁹ M ⁻¹, respectively, indicating the high and similar affinities of the three antibodies toward CEA. Radioimmunoassay binding studies of the three immunotoxins with ¹²⁵I-CEA showed that the antibody portions of the molecules retained the ability to form complexes with CEA after conjugation to ricin A chain. The maximum number of anti-CEA antibody molecules bound per cell, as demonstrated by ¹¹¹In-labeled C-19 binding assays with CEA-bearing cell lines, varied from 2.65×10⁵ per cell for HT29 to 2.01×10⁶ for LoVo, with an intermediate value of 1.17×10⁶ per cell for WiDr. Cytotoxicity of the immunotoxins was assessed by inhibition of protein synthesis and expressed as a median inhibitory dose (ID₅₀). Comparison of the ID₅₀'s of each immunotoxin on the three cell lines has shown that the immunotoxin made of the monoclonal C-19 antibody is in general 6 to 7 times more cytotoxic than the goat and baboon antibody immunotoxins. The affinity of CEA-antibody binding is probably an important, but not a sole factor in determining the immunotoxin potency. The fact that the antibodies with very similar affinity toward fluid phase CEA make immunotoxins of different potency might indicate that interactions with membrane-bound CEA are more complex and/or the efficiency of internalization of various immunotoxins is different. An important factor in immunotoxin action appears to be the CEA content in target adenocarcinoma cells.