Nanoparticle-delivered multimeric soluble CD40L DNA combined with Toll-Like Receptor agonists as a treatment for melanoma

Abstract

Stimulation of CD40 or Toll-Like Receptors (TLR) has potential for tumor immunotherapy. Combinations of CD40 and TLR stimulation can be synergistic, resulting in even stronger dendritic cell (DC) and CD8+ T cell responses. To evaluate such combinations, established B16F10 melanoma tumors were injected every other day X 5 with plasmid DNA encoding a multimeric, soluble form of CD40L (pSP-D-CD40L) either alone or combined with an agonist for TLR1/2 (Pam(3)CSK(4) ), TLR2/6 (FSL-1 and MALP2), TLR3 (polyinosinic-polycytidylic acid, poly(I:C)), TLR4 ( monophosphoryl lipid A, MPL), TLR7 (imiquimod), or TLR9 (Class B CpG phosphorothioate oligodeoxynucleotide, CpG). When used by itself, pSP-D-CD40L slowed tumor growth and prolonged survival, but did not lead to cure. Of the TLR agonists, CpG and poly(I:C) also slowed tumor growth, and the combination of these two TLR agonists was more effective than either agent alone. The triple combination of intratumoral pSP-D-CD40L + CpG + poly(I:C) markedly slowed tumor growth and prolonged survival. This treatment was associated with a reduction in intratumoral CD11c+ dendritic cells and an influx of CD8+ T cells. Since intratumoral injection of plasmid DNA does not lead to efficient transgene expression, pSP-D-CD40L was also tested with cationic polymers that form DNA-containing nanoparticles which lead to enhanced intratumoral gene expression. Intratumoral injections of pSP-D-CD40L-containing nanoparticles formed from polyethylenimine (PEI) or C32 (a novel biodegradable poly(B-amino esters) polymer) in combination with CpG + poly(I:C) had dramatic antitumor effects and frequently cured mice of B16F10 tumors. These data confirm and extend previous reports that CD40 and TLR agonists are synergistic and demonstrate that this combination of immunostimulants can significantly suppress tumor growth in mice. In addition, the enhanced effectiveness of nanoparticle formulations of DNA encoding immunostimulatory molecules such as multimeric, soluble CD40L supports the further study of this technology for tumor immunotherapy.

Figure 1. Antitumor effects of plasmids for multimeric, soluble CD40L and GITRL on established B16F10 melanoma tumors.

B16F10 cells were injected s.c. in C57BL/6 mice. When the tumors were ≥4 mm in diameter, they were injected every other day X 5 with 50 µg of plasmid DNA. Three forms of CD40L were tested as fusion proteins (see text): 1-trimer soluble CD40L (pTr-CD40L); 2-trimer CD40L (pAcrp30-CD40L); and 4-trimer CD40L (pSP-D-CD40L). Additionally, a 4-trimer form of GITRL (pSP-D-GITRL) was tested for comparison. The negative control injections were either PBS or the empty expression plasmid pcDNA3.1. Panel A – Treatment with pSP-D-CD40L or pSP-D-GITRL slowed the growth of established B16F10 tumors. Each graph shows 5 mice per group for each treatment where day 0 indicates the time when the tumor became ≥4 mm and injections began and ending when fewer than 3 mice in each group remain alive. Injections continued every other day X 5, ending on day 8 (arrows). There was a significant reduction in tumor size (mean±SEM, n = 5) compared to control pcDNA3.1 or PBS using 2-trimer pAcrp30-CD40L, 4-trimer pSP-D-CD40L, and 4-trimer pSP-D-GITRL on day 8 as measured before the final injection (p<0.05 by Student's t test). Panel B – Treatment of established B16F10 tumors with pSP-D-CD40L significantly prolonged survival. While treatment with plasmids for all 3 forms of CD40L and 4-trimer GITRL showed a trend toward enhanced survival, this was only statistically significant for the 4-trimer pSP-D-CD40L plasmid (p<0.01 by log-rank test). Consequently pSP-D-CD40L was selected for further study.

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).

Figure 3. Combinations of pSP-D-CD40L, CpG, and poly(I:C) showed strong antitumor effects on established B16F10 melanoma.

Given the promising data of Fig. 2, further studies were done to determine the relative contributions of pSP-D-CD40L, CpG, and poly(I:C) and the effects of using them in a triple combination. Twelve groups of mice (5/group) were studied in parallel. For display purposes, the data are grouped into three rows of graphs focusing on CpG (top row), poly(I:C) (middle row), and CpG + poly(I:C) (bottom row). Panels A, B, and C – While each individual agent slowed tumor growth, the most significant antitumor effect was produced by the combination of pSP-D-CD40L + CpG + poly(I:C). Panel A shows that CpG alone significantly slowed tumor growth compared to either PBS or pcDNA3.1 alone from day 12 (p<0.01 by Student's t test, mean±SEM, n = 5). In this fully controlled experiment, however, it was clear that the addition of pSP-D-CD40L to CpG produced no further antitumor effects (p>0.05). Similarly, Panel B shows that poly(I:C) alone significantly slowed tumor growth when compared to PBS or pcDNA3.1 alone from day 12 (p<0.01). Again, however, the combination of pSP-D-CD40L + poly(I:C) produced no further antitumor effects (p>0.05). Interestingly, as shown in Panel C, the double combination of CpG + poly(I:C) significantly reduced tumor growth beyond that produced by CpG alone (p<0.05 on day 24 on the combination as compared to CpG alone). The addition of pSP-D-CD40L to the two TLR agonists, CpG and poly(I:C), produced an even stronger antitumor effect (Panel C, p<0.05 on day 24 comparing the triple combination to CpG + poly(I:C)). Panels D, E, and F – For survival, the addition of pSP-D-CD40L did not increase the antitumor effects seen with CpG alone. All three agents (pSP-D-CD40L, CpG, and poly(I:C)) improved survival as single therapies. From pairwise comparisons, the survival benefit was greatest with CpG and less prominent with pSP-D-CD40L and poly(I:C). The combination of CpG + poly(I:C) improved survival further compared to poly(I:C) alone (p<0.05 by log-rank test). Although the effects on tumor growth indicated that the double combination of TLR agonists CpG + poly(I:C) was better than each alone, this was not reflected in the survival data. Similarly, the superiority of the triple combination of pSP-D-CD40L + CpG + poly(I:C) seen in the tumor growth studies was not statistically significant from the survival data.

Figure 4. Tumor-dependent differences in the immunohistology of induced tumor regression.

Panel A – Histology of control and treated tumors. Tumors were injected every other day X 5 with PBS as a control or with the triple combination of pSP-D-CD40L + CpG + poly(I:C). As shown in Figure 3, the triple combination slowed the growth of tumors, and occasionally led to tumor eradication. Two days after the last injection, tumor tissue was processed for histology by staining with hematoxylin and eosin. Tumors treated with PBS showed areas of spontaneous necrosis suggesting that the rapidly growing tumor cells often outgrow their blood supply. After treatment with the triple combination, large areas of necrotic tissue appeared containing fragmented cells and nuclear remnants consistent with a cell death process that exceeded the availability of phagocytic macrophages to clear the debris (see Panel D). Panel B – CD11c antibody staining for dendritic cells. B16F10 tumors injected with PBS as a control contained identifiable CD11c+ dendritic cells. After treatment with the triple combination, even fewer dendritic cells were found in the tumors. Panel C – CD8 antibody staining. For tumors injected with PBS as a control, relatively few CD8+ T cells were seen. However, following injections with the triple combination, there was a marked increase in intratumoral CD8+ T cells in all tumor sections examined. Panel D – F4/80 antibody staining for macrophages. Tumors injected with PBS as a control contained relatively few F4/80+ macrophages and there was no appreciable increase in F4/80+ macrophages following treatment with the triple combination.

Figure 5. PEI nanoparticle delivery of pSP-D-CD40L slowed tumor growth and prolonged survival.

The data shown are representative of three independent experiments. Panel A – Antitumor effects of PEI plasmid DNA nanoparticles prepared with pSP-D-CD40L alone or in combination with CpG or CpG + poly(I:C). The role of DNA transfection efficiency was tested by preparing nanoparticles formed from PEI and pSP-D-CD40L plasmid DNA. Intratumoral injections of PEI pSP-D-CD40L nanoparticles led to significantly slower tumor growth (p<0.05 on day 10) when compared to the injection of naked pSP-D-CD40L plasmid alone. Panel B – Survival benefit of PEI pSP-D-CD40L nanoparticle injections in combination with CpG + poly(I:C). As expected from the tumor growth data, pSP-D-CD40L formulated with PEI was able to enhance mouse survival when combined with CpG and poly(I:C) TLR agonists. This combination therapy resulted in long-term-tumor free survival of 2/5 mice (p<0.01 compared to pcDNA3.1)).

Figure 6. C32 nanoparticle delivery of pSP-D-CD40L slowed tumor growth and prolonged survival.

The data shown are representative of three independent experiments. Panel A – Antitumor effects of C32 nanoparticles prepared with pSP-D-CD40L plasmid vs. control pcDNA3.1 plasmid either alone or in combination with CpG or CpG + poly(I:C). The role of DNA transfection efficiency was tested by preparing nanoparticles formed from C32 and pSP-D-CD40L or C32 and control pcDNA3.1 plasmid DNA. Intratumoral injections of C32 pSP-D-CD40L nanoparticles plus CpG + poly(I:C) led to significantly slower tumor growth when compared to the injection of naked pSP-D-CD40L plasmid + CpG + poly(I:C) (p<0.01 on day 24). Panel B – Survival benefits of C32 pSP-D-CD40L nanoparticle injections in combination with CpG + poly(I:C). As expected from the tumor growth data, injections of nanoparticles formulated with C32 and pSP-D-CD40L enhanced survival when combined with CpG + poly(I:C) TLR agonists. Although this survival benefit was not significantly better than a similar combination using pcDNA3.1 nanoparticles instead of pSP-D-CD40L nanoparticles (p>0.05), it was significantly better than pSP-D-CD40L naked DNA plus CpG + poly(I:C) (p<0.01).