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 (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.

Immunomodulatory Effect of Imiquimod Through CCL22 Produced by Tumor-associated Macrophages in B16F10 Melanomas

“…Localized TLR7/8 activation induces antitumor immunity and partially suppresses B16F10 melanoma growth in vivo (4,7,11). To confirm the potential therapeutic effect of IQM, we first examined the effect of IQM on murine B16F10 melanoma growth in vivo.…”

“…Several reports have suggested that IQM may be an optimal reagent for invasive melanoma when used in combination with other therapies (3,4,7,10,11). Turza et al reported that IQM was effective in 10 cases of superficial dermal and subcutaneous metastasis of melanoma when used in combination with intralesional interleukin (IL)-2 (9).…”

Structure-dependent immunostimulatory effect of CpG oligodeoxynucleotides and their delivery system

“…102 In addition, co-delivery of polyriboinosinic-polyribocytidylic acid (poly(I:C)), an agonist of CpG and TLR3, with CD40 ligand (CD40L), has been reported to intensify the antitumor effect of CD40L. 103 When vector-expressing CD40L (pSP-D-CD40L) was directly administered to mouse tumor together with CpG ODN and poly(I:C), antitumor activity was displayed. When pSP-D-CD40L was loaded onto poly(β-amino esters), a cationic polymer, and polyethylenimine NPs, and delivered together with CpG ODN and poly(I:C), even greater antitumor effect was obtained.…”

Applications of nanotechnology for melanoma treatment, diagnosis, and theranostics

“…For example a nanoparticle with both CD40 and Toll-like receptor (TLR) stimulators was used to increase dendritic cells (DCs) and CD8-T-cells. 90 A nanoparticle combining TLR 7 and 9 ligands has also been demonstrated to exert a synergistic effect. 91 A nanoparticle made from lipopolyplexes was used to deliver melanoma antigen mRNA.…”