Glennie M J, Brennand D M, Bryden F, McBride H M, Stirpe F, Worth A T, Stevenson G T
Lymphoma Research Unit, General Hospital, Southampton, United Kingdom.
J Immunol. 1988 Nov 15;141(10):3662-70.
This work describes the successful use of bispecific F(ab' gamma)2 antibody (Ab) in combination with a ribosome-inactivating protein (RIP), saporin, for the treatment of neoplastic disease in vivo. A total of three thioether-linked F(ab' gamma)2 heterodimers were prepared, each having dual specificities for saporin and the guinea pig lymphoblastic leukemia, L2C. In all three cases specificity for the L2C cells was provided by a high affinity mouse anti-idiotype (anti-Id) mAb, whereas the antisaporin activity came from either one of two mouse mAb or an affinity-purified rabbit polyclonal Ab. In vitro studies, measuring protein synthesis, showed that all three derivatives were extremely efficient at delivering saporin to L2C cells, to the extent that addition of the rabbit Fab' gamma-containing bispecific Ab to cell culture at 1 microgram/ml increased the toxicity of saporin (50% inhibiting concentration) by close to 90,000-fold. Similarly, in leukemic guinea pigs, a small dose of saporin (10 micrograms) which by itself showed no therapeutic effect, was able to completely eradicate Id-positive tumor when given in combination with an excess of bispecific Ab. Although tumors did eventually emerge in most of these animals, immunofluorescence analysis showed that in almost all instances the escaping cells were Id- variants of the L2C. Experiments to define the optimal treatment regimen in this model showed that, although the administration of saporin and bispecific Ab at separate sites could be therapeutically effective, mixing the Ab and saporin to form immune complexes before injection did generally enhance their performance. A molar surplus of bispecific Ab in these mixtures both extended the metabolic survival of the saporin and enhanced the therapeutic performance, with molar ratios above 3:1 generally being required for optimum treatment when using saporin at 10 micrograms. Derivatives containing polyclonal antisaporin were more efficient than those containing mAb, yielding optimum therapeutic results with a molar ratio of 1.5:1 when combined with 10 micrograms saporin. These findings have shown that bispecific F(ab' gamma)2 Ab, as well as being straightforward to prepare, can also function as an extremely efficient vector for delivering cytotoxic agents such as ribosome-inactivating protein to unwanted cells in vivo.
本研究描述了双特异性F(ab'γ)2抗体（Ab）与核糖体失活蛋白（RIP）皂草素联合用于体内治疗肿瘤疾病的成功应用。共制备了三种硫醚连接的F(ab'γ)2异二聚体，每种对皂草素和豚鼠淋巴细胞白血病L2C均具有双重特异性。在所有三种情况下，对L2C细胞的特异性由高亲和力小鼠抗独特型（抗Id）单克隆抗体提供，而抗皂草素活性则来自两种小鼠单克隆抗体之一或亲和纯化的兔多克隆抗体。测量蛋白质合成的体外研究表明，所有三种衍生物在将皂草素递送至L2C细胞方面都极其有效，以至于以1微克/毫升的浓度向细胞培养物中添加含兔Fab'γ的双特异性抗体可使皂草素的毒性（50%抑制浓度）增加近90000倍。同样，在白血病豚鼠中，单独使用时无治疗效果的小剂量皂草素（10微克）与过量双特异性抗体联合使用时能够完全根除Id阳性肿瘤。尽管大多数这些动物最终确实出现了肿瘤，但免疫荧光分析表明，几乎在所有情况下，逃逸细胞都是L2C的Id变体。在该模型中确定最佳治疗方案的实验表明，尽管在不同部位给予皂草素和双特异性抗体可能具有治疗效果，但在注射前将抗体和皂草素混合形成免疫复合物通常会提高它们的性能。这些混合物中双特异性抗体的摩尔过量既延长了皂草素的代谢存活时间，又增强了治疗性能，当使用10微克皂草素时，通常需要摩尔比高于3:1才能达到最佳治疗效果。含有多克隆抗皂草素的衍生物比含有单克隆抗体的衍生物更有效，与10微克皂草素联合使用时，摩尔比为1.5:1时可产生最佳治疗效果。这些发现表明，双特异性F(ab'γ)2抗体不仅制备简单，还可作为一种极其有效的载体，用于在体内将细胞毒性剂如核糖体失活蛋白递送至不需要的细胞。
