Though there have been numerous attempts to develop immunotherapeutic agents to stimulate the immune system over the years, it is only recently that our understanding of the innate and adaptive immune systems has progressed to the point that rationally designed therapeutics can be designed and evaluated clinically. Therefore, there is now considerable interest in developing new immunotherapeutics for treatment of infectious diseases and cancer in companion animals and humans. Most of these new immunotherapeutics are designed to stimulate the innate immune system, which will be the primary focus of this proceeding.
Approaches to Activation of Innate Immunity
Many different microbial products can be shown to stimulate the innate immune system, but until recently exactly what component of the immune system was being triggered was largely unknown. However, the discovery of the Toll-like receptors (TLR) and more recently the NOD-like receptors (NLR) and their natural and synthetic agonists has stimulated greatly renewed interest in the field of immunotherapy. A recent example of a successful immunotherapeutic based on our new knowledge of TLR biology includes the CpG oligonucleotides (ODN), which are agonists for TLR9.1 These agonists have been widely evaluated for use in cancer immunotherapy and for use as vaccine adjuvants. In addition, a TLR 7/8 agonist (Aldara®) has been approved for topical use in humans for the treatment of basal cell carcinoma and genital warts. Activation of these TLR pathways triggers the release of inflammatory cytokines such as IL-12, IFN-α, IFN-γ and TNF that exert much of the anticancer and antiviral activity exhibited by these agents. CpG ODN have also been evaluated in veterinary medicine, including treatment of feline asthma.2,3
In addition, older drugs for which the mechanism of action was unknown have now begun to be re-evaluated for immunotherapy. For example, muramyl dipeptide and tripeptide was widely evaluated as a cancer immunotherapeutic (liposomal MTP) in dogs in the past.4 However, it is now known that MDP is an agonist for an intracellular receptor known as NOD2.5 Activation of the NOD pathway triggers many of the same cytokine responses as do the TLR agonists.
A number of older immune stimulants used in veterinary medicine (also known as biological response modifiers) were prepared as crude extracts from microbes or plants. These include acemannan (a carbohydrate extracted from the aloe plant), Immunoregulin (a heat-killed extract from Proprionobacterium), Regressin V (a cell wall extract from Mycobacterium), and inactivated rubeola virus. Most if not all of these immune stimulants activate multiple TLR pathways, which probably accounts for the majority of their activity. However, because many of these compounds are not fully purified, side-effects from injection may also develop. Therefore, it is desirable to use more fully purified and characterized molecules as immune stimulants.
Use of Recombinant Cytokines for Immunotherapy
An alternative to injection of microbial extracts is to inject high doses of the desired cytokines. The most widely investigated cytokines include interferon-alpha (IFN-α) and IL-2. Both have been extensively investigated in cancer immunotherapy and both IFN-α and IL-2 are approved for treatment of certain cancers in humans, including hairy cell leukemia, renal cell carcinoma, and melanoma. IL-2 has been evaluated to a limited degree in the treatment of canine cancer, but cost, toxicity, and antigenicity have largely made the use of human rIL-2 largely irrelevant.6 Recombinant human IFN-α has been and continues to be widely used for antiviral and cancer immunotherapy in both dogs and cats.7 However, though treatment may elicit transient benefits, the human IFN-α molecule is immunogenic in both species and rapidly elicits production of neutralizing antibodies after several weeks. Feline recombinant interferon-omega is available in Europe and has been reported for use in cats with viral infections, though randomized clinical trials have not been conducted.8,9
Immune Stimulants Based on Liposomes and TLR Agonists
Our group discovered several years ago that when charged liposomes were complexed with plasmid DNA or CpG ODN, the immune stimulatory properties of the nucleic acid agonists for TLR9 were greatly enhanced.10 The same effects were also observed when TLR3 agonists such as polyI:C were combined with liposomes.11 These immune stimulatory complexes have been extensively investigated for their ability to stimulate anti-cancer immunity and anti-viral immunity in mouse models.12-14 When cationic liposomes are mixed with DNA (such as non-coding plasmid DNA), complexes form, which are known as cationic liposome-nucleic acid complexes (CLDC) (Figure 1). Injection of these complexes by a variety of different routes, particularly by the i.v. or i.p. routes, elicits potent activation of innate immunity.
CLDC Immunotherapy for Treatment of Cancer in Dogs
We have evaluated the effectiveness of CLDC-based immunotherapy for treatment of cancer in dogs. In the first study, the effects of intravenous infusion of CLDC were evaluated in dogs with chemotherapy resistant lung metastases from osteosarcoma.15 Infusion of CLDC was shown to elicit rapid immune stimulation, as reflected by induction of fever and monocyte and macrophage activation. In addition, activation of NK cell activity was noted, which is important inasmuch as NK cells are the primary cell type responsible for the antitumor activity elicited by CLDC immunotherapy.10 Dogs treated with a series of 12 i.v. CLDC infusions over a 22-week period also had a significant improvement in overall survival times, compared to historical control animals.
In a second study, the anti-angiogenic effects of CLDC immunotherapy were assessed in dogs with soft tissue sarcoma.16 Dogs enrolled in the study received a series of 6 weekly i.v. infusions of CLDC immunotherapy. Tumor microvessel density was assessed in tumor biopsies taken before treatment, during treatment, and at the completion of treatment. We found that CLDC treatment resulted in objective tumor responses and a significant reduction in tumor microvessel density in half (6 of 12) of treated dogs that completed the 12-week trial. Side-effects (primarily transient fever) were similar to those reported in the first trial.
In a third recently completed study, we evaluated the effectiveness of CLDC as a vaccine adjuvant for use with an allogeneic cancer vaccine in dogs with soft tissue sarcoma.17 This study was based on results of a previous study in mice demonstrating the potent vaccine adjuvant activity of CLDC.11 In the study of 28 dogs with hemangiosarcoma, induction of significant antitumor immunity was documented based on anti-tumor antibody responses. In 13 dogs with splenic hemangiosarcoma that received the CLDC-based vaccine, overall survival times were significantly increased compared to a historical control population of dogs treated with chemotherapy.
Therefore, we conclude that CLDC immunotherapy can elicit potent antitumor activity in dogs by activation of innate immunity directly, particularly following i.v. administration. In addition, the CLDC platform also functions effectively as a vaccine adjuvant for cancer vaccines.
CLDC Immunotherapy for Treatment of Viral Infections and Chronic Rhinitis in Cats
The CLDC immunotherapeutic is extremely effective in eliciting interferon responses, including both type I and type II interferons. These interferons are important in eliciting antitumor activity, both also have potent antiviral activity. Therefore, we also investigated the use of the CLDC immunotherapeutic for treatment of viral infections. For example, CLDC immunotherapy was found to be extremely effective in eliciting IFN-α production and protecting mice from lethal infection with an arbovirus known as Punta Tora virus.13 We have also more recently found that treatment of mice with CLDC can protect from lethal infection with Venezuelan equine encephalitis virus and West Nile virus infection (unpublished data). Thus, the CLDC immunotherapeutic appears to have broad antiviral activity.
Chronic rhinitis in cats is a poorly understood syndrome that affects up to 5% of the cat population. The condition is suspected of having a viral etiology (e.g., feline herpesvirus or calicivirus), though this has never been conclusively proven. Because the condition may have a viral basis, we conducted a randomized clinical trial of CLDC immunotherapy in cats with chronic, antibiotic unresponsive rhinitis.18 In 12 cats with chronic rhinitis that received treatment with CLDC immunotherapy, administered every other week by the i.p. route for 8 treatments, we observed a significant improvement in clinical signs (sneezing, nasal discharge) in CLDC-treated cats compared to placebo treated cats (Figure 2). There was also a significant increase in CD4+ and CD8+ T cells in treated cats compared to untreated controls. These data suggest that CLDC immunotherapy may therefore be capable of reversing the immunologic abnormalities or state of chronic viral infection that may be responsible for chronic rhinitis in cats.
Use of CLDC Immunotherapy for Treatment of Other Infectious Diseases
CLDC immunotherapy has also been investigated for treatment of other chronic infections of dogs and cats. However, it is important to point out these examples represent very few patients and that properly designed randomized clinical trials have not yet been conducted to this point, though several are planned. In one case, CLDC immunotherapy was successfully used to cure a dog with disseminated Toxoplasma infection secondary to a CD4+ T cell deficiency, presumably by activation IFN-γ dependent killing of the organism by infected macrophages. Two animals with chronic Cryptococcus infection (one dog, one cat) have also been successfully treated with CLDC immunotherapy, though in the case of the dog the infection recurred once treatment was discontinued. Studies are currently underway to investigate the effectiveness of CLDC immunotherapy in cats with chronic stomatitis and in puppies with parvovirus enteritis. Thus, it appears that the CLDC platform may represent an effective new immunotherapeutic for application to a variety of different infectious diseases, including particularly chronic fungal and viral infections.
Strong and repeated activation of innate immunity may be used to treat cancer directly, or activation of innate immunity by an immunotherapeutic may be combined with an antigen or multiple antigens to produce specific vaccines. Though there are currently no potent and broadly effective immunotherapeutics on the veterinary market, the new CLDC immunotherapeutic may represent a new generation of defined and targeted immunotherapeutic that is suitable for treatment of cancer and chronic viral and fungal infections in dogs and cats.
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