Figure 2. A screen of TLR agonists showed that CpG and poly(I:C) had additional antitumor effects when combined with 4-trimer CD40L plasmid DNA.

Panels A and B – The combination of 4-trimer pSP-D-CD40L with CpG or poly(I:C), but not other TLR agonists tested, slowed the growth of established B16F10 tumors. As before, tumors that were ≥4 mm in diameter were injected with pSP-D-CD40L in combination with selected TLR agonists every other day X 5 (arrows). There were no apparent additive effects of Pam₃CSK₄ (TLR1/2), Malp2 (TLR2/6), FSL1 (TLR2/6), MPL (TLR4), and imiquimod (TLR7) (Panel A, mean±SEM, n = 5). The addition of poly(I:C) (TLR3) to pSP-D-CD40L showed a significantly stronger effect on tumor growth than pSP-D-CD40L alone from day 14 (Panel B, p<0.05 by Student's t test). CpG was clearly active when added to pSP-D-CD40L as compared to pSP-D-CD40L alone from day 14 (Panel B, p<0.01 by Student's t test). Panels C and D – The addition of CpG to pSP-D-CD40L resulted in a further survival benefit for mice with established B16F10 tumors. As expected from the tumor growth data, there was no increase in survival when Pam₃CSK₄, Malp2, FSL1, MPL, or imiquimod were added to pSP-D-CD40L treatment (Panel C). While the addition of poly(I:C) to pSP-D-CD40L showed a trend toward improved survival, this was not statistically significant when compared to pSP-D-CD40L alone (Panel D). In contrast, the addition of CpG to pSP-D-CD40L showed a clear survival benefit when compared to pSP-D-CD40L alone (Panel D, p<0.01 by log-rank test).