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.