French R R, Courtenay A E, Ingamells S, Stevenson G T, Glennie M J
Lymphoma Research Unit, Tenovus Laboratory, General Hospital, Southampton, United Kingdom.
Cancer Res. 1991 May 1;51(9):2353-61.
We report that selected combinations of two or more monoclonal bispecific F(ab')2 antibodies (BsAbs) far outperform single derivatives in the delivery of the ribosome-inactivating protein, saporin, to guinea pig L2C leukemic cells. Throughout the work, BsAbs were constructed by thioether-linking the hinges of two Fab'gamma, one from monoclonal anti-L2C-idiotype antibody and the other from anti-saporin antibody. The latter was either affinity-purified rabbit polyclonal or one of a panel of five mouse monoclonal antibodies. In vitro cytotoxicity studies showed that, though all derivatives were effective, the BsAb made with the polyclonal antibody was always 10 to 20 times more potent than those made with a monoclonal antibody in yielding 50% inhibition of [3H]leucine uptake. This superior activity could be matched by selective mixtures of two or more of the monoclonal derivatives. Furthermore, in immunotherapeutic delivery of saporin to tumor, a pair of BsAbs performed significantly better than did either individually. Binding and uptake studies with radiolabeled saporin demonstrated a 20-fold increase in functional affinity when saporin was held at the cell surface by an appropriate BsAb mixture rather than by a single BsAb. In contrast, only small differences were recorded in the rate at which saporin was internalized as a result of the same maneuver. We conclude that the improved performance of combinations of BsAbs arises from their ability to provide multiple linkages between saporin molecules and cell surfaces, significantly increasing the functional affinity with which saporin is tethered to the cell, but, in this system at least, having only a minor effect on the rate at which it is internalized. Cocktails of two or more BsAbs, selected to bind to multiple epitopes on ribosome-inactivating proteins and perhaps also on unwanted cells, could provide an important new strategy in immunotherapy.
我们报告称，两种或更多种单克隆双特异性F(ab')2抗体（BsAbs）的特定组合在将核糖体失活蛋白皂草素递送至豚鼠L2C白血病细胞方面，远比单一衍生物表现出色。在整个研究过程中，BsAbs是通过硫醚连接两个Fab'γ的铰链构建而成，其中一个来自单克隆抗L2C独特型抗体，另一个来自抗皂草素抗体。后者要么是亲和纯化的兔多克隆抗体，要么是一组五种小鼠单克隆抗体之一。体外细胞毒性研究表明，尽管所有衍生物都有效，但用多克隆抗体制备的BsAb在抑制[3H]亮氨酸摄取达50%时，其效力总是比用单克隆抗体制备的BsAb高10至20倍。这种优越活性可通过两种或更多种单克隆衍生物的选择性混合物来匹配。此外，在将皂草素免疫治疗性递送至肿瘤的过程中，一对BsAbs的表现明显优于单独使用的任何一种BsAb。用放射性标记的皂草素进行的结合和摄取研究表明，当皂草素通过适当的BsAb混合物而非单一BsAb固定在细胞表面时，功能亲和力提高了20倍。相比之下，由于同样的操作，皂草素内化速率的差异很小。我们得出结论，BsAbs组合性能的改善源于它们能够在皂草素分子和细胞表面之间提供多个连接，显著增加皂草素与细胞结合的功能亲和力，但至少在这个系统中，对其内化速率的影响较小。选择与核糖体失活蛋白以及可能还有不需要的细胞上的多个表位结合的两种或更多种BsAbs的混合物，可能为免疫治疗提供一种重要的新策略。
