The invention of the intellectualization cytokine using a synthetic biological response modifier.

本研究的目的是进一步探索与功能性聚合物修饰物结合在生物活性蛋白的临床应用中的作用，并通过在体内结合来提高肿瘤坏死因子-α的治疗效果。我们合成了具有内在抗肿瘤活性的二乙烯基醚-马来酸酐共聚物(DIVEMA)偶联的肿瘤坏死因子-α，作为一种合成的生物反应调节剂。DIVEMA-TNF-α的合成可以通过添加2，3-二甲基马来酸酐(DMMAn)来控制，当pH变化时，DMMAn与氨基结合或与氨基分离。用DMMAn合成的DIVEMA-TNF-α(+)在体外的比活性几乎没有下降。然而，DIVEMA-TNF-α(-)在没有被DMMAn封闭的情况下，比活性显著降低。在静脉注射24小时后，DIVEMA-TNF-α(+)与天然的TNF-α相比，对肿瘤有显著的出血性坏死效应。注射入荷S-180实体瘤的小鼠体内。此外，在每只小鼠只有100个日本参考单位的剂量下，DIVEMA-INF-α(+)显示出一种戏剧性的抗肿瘤作用，大约是天然肿瘤坏死因子-α的100倍，并且可以在所有5只携带Meth-A实体肿瘤的小鼠中诱导完全消退，而没有任何明显的副作用。因为无论是单独的DIVEMA还是肿瘤坏死因子-α和DIVEMA的混合物都不能产生静脉注射的抗肿瘤活性。给药后，肿瘤坏死因子-α的抗肿瘤活性增强可能与DIVEMA的共价结合有关。低剂量的DIVEMA-肿瘤坏死因子-α具有显著的抗肿瘤活性。由于体内注射的肿瘤坏死因子-α剂量极低，体内内源性肿瘤坏死因子-α的浓度不受影响。因此，体内的细胞因子网络并没有被破坏。这些结果表明，DIVEMA是一种有用的聚合物修饰剂，可用于连接肿瘤坏死因子-α以提高其抗肿瘤活性。

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-INF-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.