Nanoparticle-Delivered Multimeric Soluble CD40L DNA Combined with Toll-Like Receptor Agonists as a Treatment for Melanoma

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 (Pam3CSK4 ), 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

Rapid Optimization of Gene Delivery by Parallel End-Modification of Poly(β-amino ester)s

Poly(β-amino ester)s are cationic degradable polymers that have significant potential as gene delivery vectors. Here we present a generalized method to modify poly(β-amino ester)s at the chain ends to improve their delivery performance. End-chain coupling reactions were developed so that polymers could be synthesized and tested in a high-throughput manner, without the need for purification. In this way, many structural variations at the polymer terminus could be rapidly evaluated. Endmodification of the terminal amine structure of a previously optimized poly(β-amino ester), C32, significantly enhanced its in vitro transfection efficiency. In vivo, intraperitoneal (IP) gene delivery using end-modified C32 polymers resulted in expression levels over one order of magnitude higher than unmodified C32 and jetpolyethylenimine (jet-PEI) levels in several abdominal organs. The rapid end-modification strategy presented here has led to the discovery of many effective polymers for gene delivery and may be a useful method to develop and optimize cationic polymers for gene therapy

Rapid Optimization of Gene Delivery by Parallel End-modification of Poly(ß-amino ester)s

Poly(ß-amino ester)s are cationic degradable polymers that have significant potential as gene delivery vectors. Here we present a generalized method to modify poly(ß-amino ester)s at the chain ends to improve their delivery performance. End-chain coupling reactions were developed so that polymers could be synthesized and tested in a high-throughput manner, without the need for purification. In this way, many structural variations at the polymer terminus could be rapidly evaluated. End-modification of the terminal amine structure of a previously optimized poly(ß-amino ester), C32, significantly enhanced its in vitro transfection efficiency. In vivo, intraperitoneal (IP) gene delivery using end-modified C32 polymers resulted in expression levels over one order of magnitude higher than unmodified C32 and jet-polyethylenimine (jet-PEI) levels in several abdominal organs. The rapid end-modification strategy presented here has led to the discovery of many effective polymers for gene delivery and may be a useful method to develop and optimize cationic polymers for gene therapy

Gene Delivery Properties of End-Modified Poly(ß-amino ester)s

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials

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

<p>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)).</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007334#pone-0007334-g003" target="_blank">Figure 3</a>, 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.</p

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

<p>Given the promising data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007334#pone-0007334-g002" target="_blank">Fig. 2</a>, 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.</p