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Enhanced antiproliferative action of interferon targeted by bispecific monoclonal antibodies
S S Alkan1, H Towbin, H K Hochkeppel
1Pharmaceuticals Research Division, Ciba-Geigy Limited, Basel, Switzerland.
It has previously been shown that interferon (IFN) can be coupled covalently to tumor-specific monoclonal antibodies (mAb) and that the in vitro antiviral and antiproliferative action of these IFN-mAb conjugates is superior to that of uncoupled IFN. We now report a different mode of IFN delivery, i.e., via bispecific mAbs, avoiding chemical coupling of IFN. Bispecific mAbs were prepared by cross-linking two mAbs with SPDP, mAb1 being specific for an idiotype of a hybridoma cell-surface immunoglobulin and mAb2 specific for an IFN. Alternatively, Fab' fractions of mAb1 and mAb2 were coupled by disulfide formation to produce F(ab')2. Binding capacity and specificity of both arms of the mAb conjugates were first demonstrated by a solid-phase radioimmunoassay using idiotype-positive mAb as test antigen and 125I-labeled hybrid IFN-alpha B/D. Secondly, hybridomas either idiotype positive or negative were incubated with bispecific mAbs (mAb1-mAb2 or Fab'1-Fab'2) and 125I-labeled IFN at 4 degrees C. After washing away unbound reagents, the uptake of radioactivity into cells was determined. Additionally, the antiproliferative action of cold or labeled IFN targeted via different modes was assessed by an [3H]TdR incorporation method. Results showed that bispecific mAbs could specifically deliver IFN to the target cells and also inhibit their growth in vitro. Furthermore, targeting IFN by any of the three methods, IFN-mAb, mAb1-mAb2, or Fab'1-Fab'2, enhanced its in vitro antiproliferative potency compared to IFN alone.
It has previously been shown that interferon (IFN) can be coupled covalently to tumor-specific monoclonal antibodies (mAb) and that the in vitro antiviral and antiproliferative action of these IFN-mAb conjugates is superior to that of uncoupled IFN. We now report a different mode of IFN delivery, i.e., via bispecific mAbs, avoiding chemical coupling of IFN. Bispecific mAbs were prepared by cross-linking two mAbs with SPDP, mAb1 being specific for an idiotype of a hybridoma cell-surface immunoglobulin and mAb2 specific for an IFN. Alternatively, Fab' fractions of mAb1 and mAb2 were coupled by disulfide formation to produce F(ab')2. Binding capacity and specificity of both arms of the mAb conjugates were first demonstrated by a solid-phase radioimmunoassay using idiotype-positive mAb as test antigen and 125I-labeled hybrid IFN-alpha B/D. Secondly, hybridomas either idiotype positive or negative were incubated with bispecific mAbs (mAb1-mAb2 or Fab'1-Fab'2) and 125I-labeled IFN at 4 degrees C. After washing away unbound reagents, the uptake of radioactivity into cells was determined. Additionally, the antiproliferative action of cold or labeled IFN targeted via different modes was assessed by an [3H]TdR incorporation method. Results showed that bispecific mAbs could specifically deliver IFN to the target cells and also inhibit their growth in vitro. Furthermore, targeting IFN by any of the three methods, IFN-mAb, mAb1-mAb2, or Fab'1-Fab'2, enhanced its in vitro antiproliferative potency compared to IFN alone.