Electrotransfer of IL-15/IL-15Rα Complex for the Treatment of Established Melanoma.

Gene electrotransfer (GET) is a safe, reliable, and effective method of delivering plasmid DNA (pDNA) to solid tumors. GET has been previously used to deliver interleukin-15 (IL-15) to mouse melanoma, resulting in long-term tumor regression and the survival of a percentage of treated animals after challenge. To enhance this effect, we evaluated modulating the expression levels of IL-15 and co-expressing its receptor, IL-15Rα. GET was used to deliver plasmids encoding IL-15 and IL-15Rα to established B16.F10 tumors on days 0, 4, and 7. Two delivery protocols that yielded different expression profiles were utilized. Mice that were tumor-free for 50 days were then challenged with B16.F10 cells on the opposite flank and monitored for an additional 50 days. The amount of IL-15 expressed and the presence or absence of IL-15Rα in the treated tumors did not significantly affect the tumor regression and long-term survival. Upon challenge, however, low levels of IL-15 were more protective and resulted in a greater production of anti-tumor cytokines such as IFN-γ and MIP-1ß and a greater amount of CD11b+ and CD3e+ cells infiltrating tumors. While mice with high levels of IL-15 showed CD11b+ and CD3e+ cell infiltrate, there was a substantial presence of NK cells that was absent in other treated groups. We can conclude that the level of IL-15 expressed in tumors after GET is an important determinant of the therapeutic outcome, a finding that will help us finetune this type of therapy.

Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy.

Intratumoral electroporation-mediated IL-12 gene therapy (IT-pIL12/EP) has been shown to be safe and effective in clinical trials, demonstrating systemic antitumor effects with local delivery of this potent cytokine. We recently optimized our IL-12 gene delivery platform to increase transgene expression and efficacy in preclinical models. Here we analyze the immunological changes induced with the new IT-pIL12/EP platform in both electroporated and distant, non-electroporated lesions. IT-pIL12/EP-treated tumors demonstrated rapid induction of IL-12-regulated pathways, as well as other cytokines and chemokines pathways, and upregulation of antigen presentation machinery. The distant tumors showed an increase in infiltrating lymphocytes and gene expression changes indicative of a de novo immune response in these untreated lesions. Flow cytometric analyses revealed a KLRG1hi CD8+ effector T-cell population uniquely present in mice treated with IT-pIL12/EP. Despite being highly activated, this population expressed diminished levels of PD-1 when re-exposed to antigen in the PD-L1-rich tumor. Other T-cell exhaustion markers appeared to be downregulated in concert, suggesting an orchestrated "armoring" of these effector T cells against T-cell checkpoints when primed in the presence of IL-12 in situ. These cells may represent an important mechanism by which local IL-12 gene therapy can induce a systemic antitumor immune response without the associated toxicity of systemic IL-12 exposure.

Melanoma treatment with intratumoral electroporation of tavokinogene telseplasmid (pIL-12, tavokinogene telseplasmid).

Tumors evade detection and/or clearance by the immune system via multiple mechanisms. IL-12 is a potent immunomodulatory cytokine that plays a central role in immune priming. However, systemic delivery of IL-12 can result in life-threatening toxicity and therefore has shown limited efficacy at doses that can be safely administered. We developed an electroporation technique to produce highly localized IL-12 expression within tumors leading to regression of both treated and untreated lesions in animal models and in patients with a favorable safety profile. Furthermore, intratumoral tavokinogene telseplasmid electroporation can drive cellular immune responses, converting 'cold' tumors into 'hot' tumors. Clinical trials are ongoing to determine whether intratumoral tavokinogene telseplasmid electroporation synergizes with checkpoint blockade therapy in immunologically cold tumors predicted not to respond to PD-1 antibody monotherapy.

Controlled gene delivery can enhance therapeutic outcome for cancer immune therapy for melanoma.

Effective delivery still remains a major hurdle in the development of gene based therapies. While technological advances have occurred that have improved delivery in general, there is still a need for controlled delivery in order to achieve therapeutic effects. Gene electrotransfer (GET) can be utilized to accomplish this. Careful selection of parameters used for delivery such as amplitude, duration and number of pulses as well as plasmid construct can be manipulated in order to achieve appropriate levels of local expression. Previously we have shown that direct delivery of the therapeutic cytokine, interleukin 12 (IL-12), to tumors using electrotransfer can generate local and systemic anti-tumor effects in pre-clinical and clinical studies. Using this model we hypothesized that modulating local gene expression using GET can affect therapeutic outcome. To test this, we used multiple GET protocols and plasmids to achieve varying levels of local IL-12 expression. We found that high local gene expression did not give rise to a better therapeutic outcome. This suggests the level and possibly the duration of gene expression are important in mediating the host immune response against melanoma. These data also emphasize the importance of considering the desired immune outcome of the therapy when selecting parameters for GET.

Delivery of interleukin-15 to B16 melanoma by electroporation leads to tumor regression and long-term survival.

Electroporation (EP) is a method used to physically deliver therapeutic molecules such as plasmid DNA directly to tissues. It has been used safely and successfully in clinical studies and preclinical cancer models to deliver genes to a variety of tissues. In cancer research cytokine therapy is emerging as a promising tool that can be used to boost the host response to tumor antigens. The delivery of cytokines as recombinant proteins can result in toxicity and other adverse effects; however the delivery of cytokine genes using EP has been shown to be safe and effective. Interleukin 15 (IL-15) is a cytokine that promotes the innate as well as the adaptive immune response to cancer cells and bacterial pathogens. In this study we used EP to deliver a human IL-15 plasmid (phIL-15) directly to tumors to examine its anti-cancer effects. B16.F10 melanoma tumors were induced in C57BL/6J mice and phIL-15 was delivered three times over the course of a week. Expression of the transgene, tumor volume, long-term survival and resistance to challenge were monitored in these animals. Delivery of IL-15 plasmid by EP resulted in increased IL-15 expression within the tumor compared to the injection only control. This expression peaked at 12 to 18 hours after the first delivery and was sustained at lower levels after the second and third deliveries. The delivery of the phIL-15 resulted in tumor regression, long-term survival and greater protection against tumor recurrence when cancer cells were reintroduced compared to control plasmid. From these results we can conclude that the delivery of IL-15 plasmid to tumors using EP is a promising avenue to investigate for its anti-tumor effects, however more work needs to be done to increase the stability of the gene once it is delivered and to elucidate the anti-tumor mechanism.

Electroporation based gene therapy--from the bench to the bedside.

A critical aspect of gene transfer is effective delivery of the transgene to the appropriate target. Electrically mediated delivery (electroporation) of plasmid DNA has been accepted as a viable approach to achieve effective delivery. One promising area is delivering plasmid DNA to skin. Gene transfer to the skin with electroporation is currently being evaluated for its potential for inducing angiogenesis for wound healing and for delivering DNA vaccines to the skin. Experiments utilizing a plasmid encoding for vascular endothelial growth factor has demonstrated how wound healing could be accelerated. In another study, delivery of a plasmid encoding Hepatitis B surface antigen have demonstrated that high antibody titers can be induced after two applications (prime/boost). Our laboratory has also examined the use of electroporation to delivery plasmid DNA encoding various cytokines as a potential therapy for melanoma. The plasmid is injected directly into the tumor followed by the administration of electroporation. Extensive preclinical work provided the rationale for a Phase I proof of concept first in human trial in patients with accessible cutaneous melanoma metastases. Biopsies of treated lesions showed significant necrosis of melanoma cells within the tumor as well as IL-12 expression. Lymphocytic infiltrate was observed in biopsies from patients in several cohorts. Clinical evidence of responses in untreated lesions suggested there was a systemic response following therapy was observed. Since this trial several other clinical studies utilizing electroporation to deliver plasmid DNA have been initiated. It is clear that this delivery approach has tremendous potential to facilitate the translation of gene transfer protocols from the bench to the bedside.

Curcumin prevents human dendritic cell response to immune stimulants.

Curcumin, a compound found in the Indian spice turmeric, has anti-inflammatory and immunomodulatory properties, though the mechanism remains unclear. Dendritic cells (DCs) are important to generating an immune response and the effect of curcumin on human DCs has not been explored. The role curcumin in the DC response to bacterial and viral infection was investigated in vitro using LPS and Poly I:C as models of infection. CD14(+) monocytes, isolated from human peripheral blood, were cultured in GM-CSF- and IL-4-supplemented medium to generate immature DCs. Cultures were incubated with curcumin, stimulated with LPS or Poly I:C and functional assays were performed. Curcumin prevents DCs from responding to immunostimulants and inducing CD4(+) T cell proliferation by blocking maturation marker, cytokine and chemokine expression and reducing both migration and endocytosis. These data suggest a therapeutic role for curcumin as an immune suppressant.

Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained gene delivery.

PURPOSE: Thiolated chitosan appears to possess enhanced mucoadhesiveness and cell penetration properties, however, its potential in gene-drug delivery remains unknown. Herein, we report on a highly effective gene delivery system utilizing a 33-kDa thiol-modified chitosan derivative. METHODS: Thiolated chitosan was prepared by the reaction with thioglycolic acid. Nanocomplexes of unmodified chitosan or thiolated chitosan with plasmid DNA encoding green fluorescenct protein (GFP) were characterized for their size, zeta potential, their ability to bind and protect plasmid DNA from degradation. The transfection efficiency of thiolated chitosan and sustained gene expression were evaluated in various cell lines in vitro and in Balb/c mice in vivo. RESULTS: Thiolated chitosan-DNA nanocomplexes ranged in size from 75 to 120 nm in diameter and from +2.3 to 19.7 mV in zeta potential, depending on the weight ratio of chitosan to DNA. Thiolated chitosan, CSH360, exhibited effective physical stability and protection against DNase I digestion at a weight ratio>or=2.5:1. CSH360/DNA nanocomplexes induced significantly (P<0.01) higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulphide-crosslinked CSH360/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Also, intranasal administration of crosslinked CSH360/DNA nanocomplexes to mice yielded gene expression that lasted for at least 14 days. CONCLUSIONS: Thiolated chitosans condense pDNA to form nanocomplexes, which exhibit a significantly higher gene transfer potential and sustained gene expression upon crosslinking, indicating their great potential for gene therapy and tissue engineering.

Thiolated chitosan nanoparticles enhance anti-inflammatory effects of intranasally delivered theophylline.

BACKGROUND: Chitosan, a polymer derived from chitin, has been used for nasal drug delivery because of its biocompatibility, biodegradability and bioadhesiveness. Theophylline is a drug that reduces the inflammatory effects of allergic asthma but is difficult to administer at an appropriate dosage without causing adverse side effects. It was hypothesized that adsorption of theophylline to chitosan nanoparticles modified by the addition of thiol groups would improve theophylline absorption by the bronchial epithelium and enhance its anti-inflammatory effects. OBJECTIVES: We sought to develop an improved drug-delivery matrix for theophylline based on thiolated chitosan, and to investigate whether thiolated chitosan nanoparticles (TCNs) can enhance theophylline's capacity to alleviate allergic asthma. METHODS: A mouse model of allergic asthma was used to test the effects of theophylline in vivo. BALB/c mice were sensitized to ovalbumin (OVA) and OVA-challenged to produce an inflammatory allergic condition. They were then treated intranasally with theophylline alone, chitosan nanoparticles alone or theophylline adsorbed to TCNs. The effects of theophylline on cellular infiltration in bronchoalveolar lavage (BAL) fluid, histopathology of lung sections, and apoptosis of lung cells were investigated to determine the effectiveness of TCNs as a drug-delivery vehicle for theophylline. RESULTS: Theophylline alone exerts a moderate anti-inflammatory effect, as evidenced by the decrease in eosinophils in BAL fluid, the reduction of bronchial damage, inhibition of mucus hypersecretion and increased apoptosis of lung cells. The effects of theophylline were significantly enhanced when the drug was delivered by TCNs. CONCLUSION: Intranasal delivery of theophylline complexed with TCNs augmented the anti-inflammatory effects of the drug compared to theophylline administered alone in a mouse model of allergic asthma. The beneficial effects of theophylline in treating asthma may be enhanced through the use of this novel drug delivery system.

Immunobiology of grass pollen allergens.

Among pollen allergens, grass pollen allergens are some of the most frequent contributors to allergic symptoms. Substantial progress has been made since the 1960s in the identification and characterization of the grass allergens. Members of this group belong to the Poaceae family, and have been classified into 13 distinct groups based on their structure, and their biological and immunologic properties. The major contributors to allergy and, hence, most studied among the grass allergens, are those belonging to groups 1 and 5. This review is focused on the structure and immunobiology of the grass allergens and highlights how recent advances in the field have contributed to superior diagnosis and immunotherapy for allergy to grass pollens.