Specific enzyme-linked immunosorbent assays for quantitation of antibody-cytokine fusion proteins

Preliminary testing has shown in vitro and in vivo that antitumor activity can be obtained with fusion proteins linking tumor-reactive monoclonal anti bodies to cytokines, such as granulocyte macrophage colony-stimulating fact or or interleukin 2 (IL-2), Preclinical and clinical testing of these reage nts requires their in vitro and in vivo quantitation and pharmacokinetic ev aluation. We have focused on the detection of a fusion protein which links one human IL-2 molecule to the carboxy terminus of each heavy chain of the tumor-reactive human-mouse chimeric anti-GD2 antibody, ch14.18. We have dev eloped enzyme-linked immunosorbent assays (ELISAs) to evaluate intact tumor -reactive fusion proteins, By these ELISAs we can reliably measure nanogram quantities of intact ch14.18-IL-2 fusion protein and distinguish the intac t protein from its components (ch14.18 and IL-2) in buffer, mouse serum, an d human serum with specificity and reproducibility, The measurement of inta ct ch14.18-IL-2 fusion protein is not confounded by free IL-2 or free ch14. 18 when 100 ng or less of total immunoglobulin per mi is used during the as say procedure. Our results indicate that these ELISAs are suitable for prec linical and clinical testing and with slight modifications are applicable t o the analysis of a variety of other fusion proteins.