Fig. 4. Biological activity of IPR8-GMBS-R848 conjugates made by 2-step and 1-step method.
RAW 264.7 cells were treated with IPR8-SM(PEG)4-R848, IPR8-GMBS-R848, IPR8-ML2-103-1 or IPR8 for 24 hrs. CD40 and TNFα production were measured as indicators of activation. (a) Representative histogram showing CD40 production after a 24 hr treatment with IPR8 conjugates or IPR8 alone at a dose of 1.1 μg/ml. Data shown in (b) CD40 and (c) TNFα production depict the mean ± SEM from at least 3 experiments. The gray square depicts no stimulation, in which no TNFα was detected. Statistical significance was determined by unpaired Student’s t test. * p<0.05 for the indicated doses. ** p< 0.005 for the indicated doses. (d) Quantitation of live virus-associated free thiols before and after conjugation of R848. A commercial fluorescence-based assay for free thiol content was used as an indirect measure of the amount of R848 bound to PR8 before and after completing the conjugation protocol. Fluorescence units were normalized to protein content of unconjugated PR8, PR8-SM(PEG)4-R848 and PR8-GMBS-R848 virus conjugates. For the unconjugated PR8 control, DMSO vehicle was added in place of R848-linker compounds and subject to the same protocol steps as for R848-conjugated PR8. The mean±SD from 3 independent experiments is shown. There was no statistical difference (ns, p>0.05) between PR8-SM(PEG)4-R848 and PR8-GMBS-R848 by one-way ANOVA with Tukey’s multiple comparisons post-test. Differences between PR8 and each R848 conjugate were significant (****p < 0.0001).