TITRATION CALORIMETRY STUDY OF AN ANTIIDIOTYPIC ANTIBODY CASCADE IN AHUMAN MELANOMA-ASSOCIATED ANTIGEN SYSTEM

The thermodynamic parameters of interactions between six variants of t he anti-idiotypic monoclonal antibody (mAb) CGP 60686 produced by the hybridoma MK2-23 with an idiotypic mAb and five different anti-anti-id iotypic mAb were studied with high sensitivity titration calorimetry. CGP 60686 recognizes an epitope in the antigen-combining region of the human high-molecular-weight-melanoma-associated antigen (HMW-MAA)-spe cific mouse mAb CGP 76873 produced by the hybridoma 763.74. The five H MW-MAA-specific anti-anti-idiotypic mAbs GH 464, GH 518, GH 149, GH 38 6 and GH 586 were generated from mice immunization with mAb CGP 60686. All interactions between the anti-idiotypic mAb and the idiotypic mAb or the anti-anti-idiotypic mAb showed large exothermic binding enthal pies between -15 and -23 kcal/mol and binding affinities larger than 6 x 10(9) M-1. Four of the five anti-anti-idiotypic mAbs tested exhibit ed significantly higher binding enthalpies for the interaction with th e anti-idiotypic than the idiotypic mAbs. Replacement of either the he avy or the light chain variable region of the anti-idiotypic mAbs with an unrelated variable region abolished the idiotype to anti-idiotype interaction. Thus, both the heavy and the light chain variable region of the anti-idiotypic mAbs are required for binding to the idiotype. T he values of the binding enthalpy showed only small variations when bi nding of the idiotypic mAb CGP 76873 to four variants of the anti-idio typic mAb CGP 60686 with different immunoglobulin constant regions, bu t identical variable regions were compared. Furthermore, Fab fragments of the idiotypic mAbs showed almost the same binding enthalpy per bin ding site as the whole IgG molecules. Immunoglobulin constant regions thus had little influence on the idiotype to anti-idiotype interaction s. Taken together, the observed thermodynamic parameters suggest that the idiotype to anti-idiotype interactions studied here are enthalpy-d riven processes with only minor entropic contributions. High sensitivi ty titration calorimetry was used to monitor protein-protein interacti ons within an antiidiotypic antibody cascade. It was found that the di rect measurement of the interaction enthalpy allowed st quantitative c haracterization of the binding processes studied. (C) 1997 Elsevier Sc ience Ltd. All rights reserved.