(D), Viability of MCF-7HER2 cells in the presence of different amounts of fetal bovine serum and 1.5 μM F-Ade was determined after 72 hours of incubation by MTS assay. Error bars for each graph represent standard Doramapimod molecular weight deviation within each set of values. Conversion of F-dAdo to F-Ade by cell bound hDM-αH-C6.5 MH3B1 results in bystander activity For ADEPT to be effective, the cytotoxic drug generated

by the activity of the cell associated enzyme should be cytotoxic to the neighboring cells that may lack the expression of the tumor associated antigen. To investigate the bystander effect of F-Ade generated by the enzymatic activity of hDM-αH-C6.5 MH3B1, different ratios of CT26HER2/neu and CT26 cells were mixed and seeded. The next day, cells were incubated with 0.1 μM of hDM-αH-C6.5 MH3B1 for 45 minutes, washed twice, and after 72 hours the level of inhibition of cell proliferation caused by F-Ade that was generated by the enzymatic activity of bound hDM-αH-C6.5 MH3B1 was determined by MTS assay. Complete inhibition of cell proliferation was achieved when up to 35% of the seeded cells were comprised of CT26 (Fig. 5B). When 75% of the cells were CT26, 50% inhibition of cell growth was observed (Fig. 5B). This result indicates that the F-Ade generated by the enzymatic activity KPT-330 of hDM-αH-C6.5 MH3B1 bound to CT26HER2/neu is not only toxic to HER2/neu expressing cells, but also to the neighboring cells that lack the expression of tumor

antigen. F-Ade is toxic to rapidly, slowly and non-dividing cells Since it has been shown that the non-dividing stromal cells play a critical role in providing support for tumor growth, and since tumors are composed of cells growing at different rates, we examined the cytotoxic affect of F-Ade on slowly-dividing or non-dividing cells. MCF-7HER2 cells were grown overnight in growth medium that contained 10% fetal bovine serum. The next day, cells were washed and incubated for 72 hours in medium that contained varying amounts of serum. MCF-7HER2 cells divided even with serum levels as low as 0.25% and ceased to divide, but

remained viable only when no serum was present (Fig. 5C). In the presence of different concentrations of F-Ade, similar cytotoxicity was observed irrespective of the rate of cell growth (Fig. 5D). This indicates that F-Ade is toxic to the rapidly or slowly growing tumor cells as well as to the non-dividing Phospholipase D1 neighboring cells that may sustain tumor growth. Novel MHCII binding peptides present in hDM-αH-C6 MH3B1 B cells are activated to develop into antibody producing plasma cells when their B cell receptor interacts with non-self epitopes on soluble proteins and when they receive a signal from TH cells. It seems likely that hDM-αH-C6 MH3B1 will exhibit minimal reactivity with the B cell receptor because the two introduced mutations are buried within the purine binding pocket of hDM and the structure of hDM is extremely similar to the structure of wild type enzyme [13].