The purpose of this study is to further explore the usefulness of conjugation with functional polymeric modifiers for clinical application of bioactive
proteins and to increase the therapeutic efficacy of
tumor necrosis factor alpha (
TNF-alpha) by conjugation in vivo. We synthesized
TNF-alpha conjugated with the copolymer of
divinyl ether and
maleic anhydride (
DIVEMA), which has intrinsic antitumor activity as a synthetic
biological response modifier. The synthesis of
DIVEMA-
TNF-alpha could be controlled by the addition of
2,3-dimethylmaleic anhydride (DMMAn), which binds to or separates from amino groups when the pH is changed. The specific activity of
DIVEMA-
TNF-alpha (+) synthesized with DMMAn was hardly decreased in vitro. However,
DIVEMA-
TNF-alpha (-), which is conjugated without blocking by DMMAn, had a markedly diminished specific activity.
DIVEMA-
TNF-alpha (+) caused a dramatic hemorrhagic necrotic effect on the
tumor when compared to native
TNF-alpha 24 h after i.v. injection into mice bearing Sarcoma-180 solid
tumors. In addition,
DIVEMA-
TNF-alpha (+) at a dose of only 100 Japan reference units per mouse revealed a dramatic antitumor effect that is approximately 100 times greater than native
TNF-alpha and that could induce complete regression in all five mice bearing Meth-A solid
tumors without any apparent side effects. Because neither
DIVEMA alone nor a mixture of
TNF-alpha and
DIVEMA caused antitumor activity with i.v. administration, the increase in antitumor potency of
TNF-alpha may be caused by the covalent conjugation with
DIVEMA.
DIVEMA-
TNF-alpha at low dose revealed dramatic antitumor potency. Because
TNF-alpha injected in vivo is extremely low-dose, concentration of intrinsic
TNF-alpha in vivo is not influenced. Therefore, the
cytokine network in vivo is not destroyed. These results suggest that
DIVEMA is a useful polymeric modifier for conjugation of
TNF-alpha to increase its antitumor activity.