Figure 10.
The effects of the FabH (Her2)‐linker‐GA1‐FabLRT(OKT3/UTCH1) BiTE on PBMC/SKBR3 (10:1) co‐cultures. To test the effect of the BiTE, 20K SKBR3 cells were cultured on a plate overnight. 200K of PBMCs were mixed with 50 nM of the BiTE and added on the SKBR3 cells. Cell killing effect measured by LDH activity (a) and cytokine release upon T cell activation (b, c) were measured after 24 hr incubation. As a control, all the individual components of the BiTE reagents (lanes 1 and 2) and with mutant CD3 FabLRT, deficient in CD3 binding (lanes 4 and 6) were tested and showed practically no effect on LDH or cytokine levels (dashed line). The CD3 activation and cell killing was observed only when both active components of the BiTE were present (lanes 3, 4, 7, and 8) and with genetically linked bi‐specific molecule used as a positive control for the immunological synapse formation 9. Results of representative experiments out of three (or more) are shown. Contents of lanes: 1 (GA1+ FabH(Her2) + FabLRT(OKT3); 2 (GA1+ FabH(Her2) + FabLRT(UTCH1), 3 (FabH(Her2) + GA1 + FabLRT(OKT3); 4 (FabH(Her2) + GA1 + mutFabLRT(OKT3)); 5 (FabH(Her2) + GA1 + FabLRT(UTCH1); 6 (FabH(Her2) + GA1+ mutFabLRT(UTCH1); 7 (FabH(OKT3) + GA1+ FabLRT(Her2); 8 FabH(UTCH1) + GA1+ FabLRT(Her2); 9 “BiTE control”: FabH(OKT3) fused to Her2 scFV