CYTOKINE TARGETING IN TUMORS USING A BISPECIFIC ANTIBODY-DIRECTED AGAINST CARCINOEMBRYONIC ANTIGEN AND TUMOR-NECROSIS-FACTOR-ALPHA

The use of tumor necrosis factor alpha (TNF alpha) in cancer therapy i s limited bg its short circulatory half-life and its severe systemic s ide effects. To overcome these limitations, we evaluated the capabilit y of a bispecific antibody (BAb) directed against carcinoembryonic ant igen (CEA) and human TNF alpha to target this cytokine in tumors. A BA b was constructed by coupling the Fab' fragments from an anti-CEA mono clonal antibody (MAb) to the Fab' fragments from an anti-TNF alpha MAb via a stable thioether linkage. The double specificity of the BAb for CEA and TNF alpha was demonstrated using a BIAcore(TM) two-step analy sis. The affinity constants of the BAb far CEA immobilized on a sensor chip and for soluble TNF alpha added to the CEA-BAb complex were as h igh as those of the parental MAbs (1.7 X 10(9) M(-1) and 6.6 X 10(8) M (-1), respectively). The radiolabeled I-125-labeled BAb retained high immunoreactivity with both CEA and TNF alpha immobilized on a solid ph ase. In nude mice xenografted with the human colorectal carcinoma T380 , the I-125-labeled BAb showed a tumor localization and biodistributio n comparable to that of I-131-labeled anti-CEA parental F(ab')(2) with 25-30% of the injected dose (ID)/g tumor at 24 h and 20% ID/g tumor a t 48 h. To target TNF alpha to the tumor, a two-step i.v. injection pr otocol was used first, in which a variable dose of I-125-labeled BAb w as injected, followed 24 or 48 h later by a constant dose of I-131-lab eled TNF alpha (1 mu g). Mice pretreated with 3 mu g of BAb and sacrif iced 2, 4, 6 or 8 h after the injection of TNF alpha showed a 1.5- to 2-fold increased concentration of I-131-labeled TNF alpha in the tumor as compared to control mice, which received TNF alpha alone. With a h igher dose of BAb (25 mu g), mice showed a better targeting of TNF alp ha with a 3.2-fold increased concentration of I-131-labeled TNF alpha in the tumor: 9.3% versus 2.9% ID/g in control mice 6 h after TNF alph a injection. In a one-step injection protocol using a premixed BAb-TNF alpha preparation, similar results were obtained 6 h postinjection (3 .5-fold increased TNF alpha tumor concentration). A longer retention t ime of TNF alpha was observed leading to an 8.1-fold increased concent ration of TNF alpha in the tumor 14 h postinjection (4.4 versus 0.5% I D/g tumor for BAb-treated and control mice, respectively). These resul ts show that our BAb is able, first, to localize in a human colon carc inoma and, there, to immunoabsorb the i.v.-injected TNF alpha, leading to its increased concentration at the tumor site.