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.