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

Pre-Clinical Evaluation of a Replication-Competent Recombinant Adenovirus Serotype 4 Vaccine Expressing Influenza H5 Hemagglutinin

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine

EBV LMP1, a viral mimic of CD40, activates dendritic cells and functions as a molecular adjuvant when incorporated into an HIV vaccine

LMP1, a constitutively active analog of CD40, is shown to function as molecular activator of human dendritic cells and macrophages that can be used as a vaccine adjuvant. HIV-1 does not significantly activate cellular immunity, which has made it difficult to use attenuated forms of HIV-1 as a vaccine. In contrast, EBV induces robust T cell responses in most infected individuals, perhaps as this virus contains LMP1, a viral mimic of CD40, which is a key activating molecule for DCs and macrophages. Consequently, studies were conducted using LMP1 and LMP1-CD40, a related construct formed by replacing the intracellular signaling domain of LMP1 with that of CD40. Upon electroporation into DCs, LMP1 and LMP1-CD40 mRNAs were sufficient to up-regulate costimulatory molecules and proinflammatory cytokines, indicating that these molecules can function in isolation as adjuvant-like molecules. As a first step toward an improved HIV vaccine, LMP1 and LMP1-CD40 were introduced into a HIV-1 construct to produce virions encoding these proteins. Transduction of DCs and macrophages with these viruses induced morphological changes and up-regulated costimulatory molecules and cytokine production by these cells. HIV-LMP1 enhanced the antigen-presenting function of DCs, as measured in an in vitro immunization assay. Taken together, these data show that LMP1 and LMP1-CD40 are portable gene cassettes with strong adjuvant properties that can be introduced into viruses such as HIV, which by themselves, are insufficient to induce protective cellular immunity

Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

► Multimeric soluble BAFF, 4-1BBL, LIGHT, and CD70 can adjuvant HIV-1 DNA vaccines. ► CD40L, LIGHT, OX40L, and 4-1BBL enhance memory T cell avidity and IL-2 secretion. ► The B cell costimulator BAFF is unable to increase antibody levels in a DNA vaccine. DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP-D-OX40L and SP-D-CD70 increased T H1 (IgG2a) but not T H2 (IgG1) antibody responses in the vaccinated animals. Surprisingly, the B cell-activating protein BAFF did not enhance anti-Gag antibody responses when given as an SP-D fusion adjuvant, but nonetheless enhanced CD4+ and CD8+ T cell responses. We present evidence that various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid. These data support the continued evaluation of SP-D-TNFSFL fusion proteins as molecular adjuvants for DNA and/or viral vector vaccines. Constructs of particular interest included SP-D-OX40L, SP-D-4-1BBL, SP-D-LIGHT, and SP-D-CD70. SP-D-BAFF was surprisingly effective at enhancing T cell responses, despite its inability to enhance anti-Gag antibody secretion

Macaque multimeric soluble CD40 ligand and GITR ligand constructs are immunostimulatory molecules in vitro.

CD40 ligand (CD40L) and GITR ligand (glucocorticoid-induced tumor necrosis factor receptor-related protein ligand [GITRL]) are tumor necrosis factor superfamily molecules that can be used as vaccine adjuvants. In a previous human immunodeficiency virus (HIV) DNA vaccine study in mice, we found that plasmids expressing multimeric soluble forms of trimeric CD40L (i.e., many trimers) were stronger activators of CD8(+) T-cell responses than were single-trimer soluble forms or the natural membrane-bound molecule. This report describes similar multimeric soluble molecules that were constructed for studies in macaques. Both two-trimer and four-trimer forms of macaque CD40L were active in B-cell proliferation assays using macaque and human cells. With human cells, four-trimer macaque GITRL costimulated CD4(+) T-cell proliferation and abrogated the immunosuppressive effects of CD4(+) CD25(+) regulatory T cells on a mixed leukocyte reaction. These molecular adjuvants provide new tools for vaccine development in the simian immunodeficiency virus system and other macaque models

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

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

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

Regression of Established AB1 Murine Mesothelioma Induced by Peritumoral Injections of CpG Oligodeoxynucleotide Either Alone or in Combination with Poly(I:C) and CD40 Ligand Plasmid DNA

Stimulation of the CD40 receptor using an agonistic anti-CD40 antibody can slow the growth of AB1 tumors. Stimulation of the GITR receptor may also have antitumor activity by countering the immunosuppressive effects of regulatory CD4+ T cells. Similarly, agonists for Toll-Like Receptors (TLR) such as CpG oligodeoxynucleotides (TLR9 agonist) have activity against AB1 tumors. Combinations of CpG with CD40 ligand and polyinosinic-polycytidylic acid (poly(I:C), TLR3 agonist) may be even stronger than CpG alone. The synergistic effects of these combinations have been tested in other tumor types but not in mesothelioma. Established AB1 mesothelioma tumors were injected with either plasmid DNA encoding a novel 4-trimer form of murine CD40 ligand (pSP-D-CD40L), GITR ligand (GITRL), or control plasmid DNA. In addition, CpG with or without poly(I:C) was also injected intratumorally. Plasmid injections of pSP-D-CD40L or pSP-D-GITRL, had no significant antitumor effect, possibly reflecting the difficulty of administering DNA injections into this very dense tissue. However, the injection of CpG with or without poly(I:C) strongly suppressed tumor growth and led to long-term tumor-free survival. The response to a triple combination of pSP-D-CD40L + CpG + poly(I:C) was demonstrated by an increase in intratumoral CD8+ T cells and a dramatic increase in F4/80+ macrophages. Intratumoral injections of plasmid DNAs encoding highly active forms of either CD40 ligand or GITR ligand had no significant antitumor effects in this model, although improved DNA delivery techniques could possibly improve this strategy. In contrast, intratumoral CpG injections had significant antitumor effects and there were indications that CpG plus poly(I:C) was even more effective. Taken together, these data confirm previous reports that immune stimulants, especially CpG TLR9 agonists, have potential as a treatment for mesothelioma