The New Immunotherapy for Cancer: What You Need to Know

The New Immunotherapy for Cancer: What You Need to Know Barbara E. Kitchell, DVM, Ph.D., DACVIM VCA Veterinary Care Animal Hospital and Referral Center Albuquerque, NM The immune system is a miraculous and wonderful thing, capable of protecting us from many of life's little (or big) annoyances such as viral and bacterial diseases. Any foreign agent that can be recognized by the immune system is antigenic, and if the immune system can mount an active response against the foreign agent, it is immunogenic. Not all antigens are immunogenic, and herein lies the difficulty of cancer immunotherapy. Many investigators believe that the immune system has a major function in immune surveillance against cancer development. In the view of these investigators, the patient who develops overt malignancy has suffered an immune system failure. tii s true that a large body of evidence has been amassed over the years demonstrating that manipulation of the immune response can lead to tumor regression and occasionally to cure. Most of this work has been performed with rodents bearing transplantable tumors, where the antigenic difference between the tumor and host cells is large. Unfortunately, there is less compelling evidence for the reliable therapeutic benefits of the immune system in spontaneous animal and human malignancies. In order for the immune system to kill cancer cells, target molecules must be present that are immunogenic. These target molecules should be specific to the tumor in order for a differential response to be mounted against it. The presence of tumor associated antigens (TAA) is supported by research in rodents with transplantable, virally induced and chemically induced tumor models. In the case of long established transplantable tumors in rodents, the immune system is capable of eradicating modest tumor burdens, and the treated animal is protected against later challenge with the same tumor cells. Unfortunately, it is thought that although the transplantable tumors were originally syngeneic with the host mice into which they are implanted, over the course of many cell culture and animal passages, the cells experience genetic drift, or mutations which allow the immune system to "see" them. Tumors that are induced in rodents by viruses or high doses of carcinogens are usually highly immunogenic, but chronic low dose administration of carcinogens (the most likely model for human and spontaneous animal tumor development) produces tumors that are weakly immunogenic at best. In short, cancers that arise in an individual should not trigger the immune system; they are not foreign in origin and thus are tolerated, at least above a certain threshold of cell dose. If immune surveillance is useful to the body, it is in scavenging out micrometastatic disease

or very tiny early malignancies. Immunotherapy - There are several approaches to immunotherapy that have been tried. They are characterized as active, passive, specific and non-specific. In the case of active immunotherapy, attempts are made to stimulate the immune response of the host by "firing up" the immune system by nonspecific means such as injections of BCG, or newer analogs like Immunocidin, or by specific means such as injections of killed, modified tumor cells (autologous vaccine approach). Passive immunity refers to the transfer of immune effectors (cells or antibodies) from an immunized individual into the cancer-bearing patient. Examples of these approaches include serotherapy (injection of immune serum for immunized animals), monoclonal antibody injections, or adoptive immunotherapy with injection of effector cells (tumor infiltrating lymphocytes, lymphokine activated killer cells, or natural killer cells) from immunized animals. Newer non-specific active immune approaches include the injection of recombinant proteins that act as biologic response modifiers such as Interleukin-2, Tumor Necrosis Factor, and Interferons to stimulate an immune response. Overcoming immune tolerance of cancer is a highly desirable yet major challenge to effective cancer therapy. Numerous strategies have been proposed with the common goal of stimulating cell-mediated immune responses to cancer. Cell-mediated immunity may benefit cancer patients in several ways including the generation of tumor reactive lymphocytes capable of killing cancer cells, systemic circulation of tumoricidal lymphocytes with the capacity to kill metastatic foci, potent killer immune cells able to destroy chemotherapy-resistant tumor cells, and the development of long-term endogenous tumor surveillance through the generation of tumor-specific memory T cells. Head and neck cancer, and canine oral melanoma in particular, has become the "testing ground" to explore and develop some strategies for immunotherapy. With a few exceptions in veterinary medicine, at this time all efforts to harness the immune system It combat cancer remain investigational and there are few biological response modifiers that are routinely available in private practice. One notable exception to this statement is the xenogeneic melanoma vaccine (Oncept) from Merial, which is advancing the field of veterinary immunotherapy and helping patients live longer, healthier lives, at least in theory. More recently, a mycobacterial cell wall fraction has been commercialized for treatment of mixed mammary tumors and mammary adenocarcinoma in dogs, (Immunocidin). It may be premature to consider immunotherapy as the "best way" to treat cancer now, but the intense research, novel approaches, and achievements to date herald a bright and promising future for this discipline. Immunotherapy for melanoma - Long-term survival of dogs with advanced malignant melanoma was achieved after DNA vaccination with xenogeneic human tyrosinase antigen, available under limited license in the United States from Merial (Oncept ). This xenogeneic vaccine contains human DNA for the gene tyrosinase, which is highly expressed in melanocytes as part of the production of melanin pigment. The gene is under the control of a strong cytomegalovirus (CMV) promoter, which results in production of the human protein in canine muscle. The vaccine is introduced to muscle by use of a needle-less bioinjector system. Once the dog begins to produce the human

protein, the canine immune system recognizes the human tyrosinase enzyme and is hopefully sufficiently potent to break self-tolerance to eradicate remaining microscopic (or in some cases even macroscopic) melanoma tumor burden in the dog. The vaccine is administered on an every other week basis for 4 treatments, then as booster injections every 6 months. Responses have been seen even in dogs with transient tumor progression. Optimally, dogs should have the tumor burden reduced to T0 (microscopic) by surgery and/or radiation therapy prior to vaccination. In the phase one trial, median survival for 9 dogs treated was 389 days, with complete responses reported and greater that 588-day survival for one dog with bulky non-resectable disease. Dogs that demonstrated antigen specific antibody responses were more likely to have positive responses clinically. Expanded trials have been reported in abstract form but limited peer-reviewed publications are available for evaluation. However, these expanded trials were sufficient to result in full licensure of the product by the FDA in 2010. Other immune approaches under study for canine oral melanoma include treatment with allogeneic and autologous tumor cell vaccines, gene therapy, cytokines, and antibodies. The rationale for tumor cell vaccines is based on the idea that attenuated transfected tumor cells may serve as an antigen source and a vehicle to deliver immunostimulatory cytokines concurrently. Following surgery to remove all gross melanoma, the gene for GM-CSF is transfected into the dog's own tumor cells (autologous) or canine melanoma cells from an established cell line (allogeneic) and these cells are injected repeatedly to induce antitumor immunity. It is a well-tolerated, easily administered treatment making it attractive for the practice setting. Intratumoral injection of immunostimulatory genes (e.g., interleukin-2, staphylococcal enterotoxin B) is another strategy that relies on melanoma cells to secrete immunostimulatory molecules and has also demonstrated clinical activity. Intratumoral injection of genes that promote apoptosis (e.g., Fas ligand, p21, etc.) to increase the availability of tumor antigen for immune cell processing and presentation, are under study as well. Systemic administration of interleukin-2 (IL-2) also has been shown to activate the immune system of dogs with advanced oral melanoma, and anti melanoma monoclonal antibodies (e.g., antiganglioside G02, G03) have been combined with IL-2 to promote trafficking of immune cells to tumor targets. COX-2 and Cancer-After cellular injury, lipids are released from plasma membranes and metabolized into mediators capable of changing cellular physiology. Since these lipids are present at the first site exposed to the injury, they provide an ideal substrate for the synthesis of defensive mediators and homeostatic regulators. One such group of lipid mediators is the prostanoids, including PGs and thromboxanes (TXs). Prostanoids were initially categorized as proinflammatory, but as the understanding of prostanoid physiology has evolved, it has become clear that these substances can act to both promote and inhibit inflammation. Prostanoid production depends on the activity of the two COX isoenzymes. COX-l is present in most cells and its expression is generally constitutive. In contrast, COX-2 is found in the brain and kidneys where in other organs its expression is low or undetectable, until stimulation takes place when its activity dramatically increases. Increased expression of COX-1 upon stimulation also can occur

and has been found in mast cells, macrophages and dendritic cells, in certain inflammatory responses, at the first 30 minutes after stimulation. In the same way, COX- 2 can be upregulated in special physiologic situations such as in the ovaries during ovulation and pregnancy. PGH2 is produced by both COX isoforms and is the common substrate for a series of specific synthase enzymes that produce PGD2, PGE2, PGF2- alpha, PGI2 and TXA2. In the cancer scenario, COX-2 overexpression has been associated with numerous carcinomas (eg: colon, gastric, esophageal, breast, pancreatic) as well as some sarcomas and melanoma. COX-2 effects on cell cycle have been associated with increased angiogenesis, increased cell growth, resistance to apoptosis, decreased adhesiveness, resistance to chemotherapy, and increased invasiveness. COX-2 overexpression has been associated with aggressiveness in colon, lung, SCCHN, and breast cancers. In breast cancer patients, strong association was found between expression of COX-2 and MDRl/Pgp-170 and consequently, resistance to chemotherapy. The theory that prostaglandins and NSAIDs can affect carcinogenesis arose from the relationship between chronic inflammation and cancer. Ulcerative colitis, eosinophilic cystitis, pelvic inflammatory disease, and systemic lupus erythematosis in humans, have all been associated with increased risk for malignancies. In support of this concept, studies have shown that colonic adenomatous polyps and colon cancer, can be prevented in up to 50% among patients who took aspirin for more than four years. Similar results have recently been obtained in trials that access prevention of breast cancer. Optimal frequency of administration and dose are unknown, but one study demonstrated that with infrequent aspirin use (less than once a month), the relative risk of death due to colon cancer were 0.77 for men and 0.73 for women; this risk decreased further to 0.60 for men and 0.58 for women if aspirin was taken more than 16 times per month. In dogs, overexpression of COX-2 has been observed in transitional cell carcinoma of the bladder and squamous cell carcinoma. In general NSAIDs will act on tumors by COX-2 dependent as well as COX-2 independent mechanisms. Piroxicam, a COX-nonspecific NSAID, is widely accepted as a treatment for canine transitional cell carcinoma (TCC). Mechanistically, benefits from piroxicam are attributed primarily to its ability to block COX2 activity in the cancer cells. COX2 has been associated with immunosuppressive effects of prostaglandins (e.g., PGE2) and there is growing interest in the use of COX2 inhibitors to treat human cancer. Although response rates are low, the observation that TCC responds to piroxicam at all implies canine bladder cancer is not an immunologically privileged site and may be an interesting tumor for other immune interventions. Cox-2 inhibition may also work through anti-angiogenic means as well. Metronomic chemotherapy as an anticancer strategy Targeting of chemotherapeutic agents or other substances that may inhibit endothelial cell growth (e.g., vascular endothelial growth factor receptor therapy) represent promising strategies for cancer treatment. Low dose continuous chemotherapy has the capacity to not only affect cancer cells themselves, but also to normalize a suppressed immune response. Recent studies by Dr. Barb Biller of Colorado State University

indicate that low doses of cyclophosphamide (ie 15 mg/m2 PO daily as reformulated capsules) can re-balance the immune system by decreasing levels of regulatory T lymphocytes (T-regs), which act as suppressor T s. In dogs with high levels of CD8 cytotoxic T lymphocytes compared to T-regs, survival and disease free interval was statistically prolonged in canine osteosarcoma patients when compared to dogs with a decreased CD8/T-reg ratio. Thus, people have begun using metronomic combination chemotherapy using a NSAID drug such as piroxicam, daracoxib, or meloxicam, along with continuous low dose cyclophosphamide by mouth. Some protocols are supplemented with other potentially therapeutic agents such as toceranib, which is a VEGFR2 inhibitor, or doxycycline. These studies are ongoing, but the impact of this approach is also being promoted because of the potential for cost-effective anticancer therapy. Non-Specific Active Immunotherapy There has been a long series of studies investigating agents that activate various components of the immune system as anticancer strategies. Dr. Greg MacEwen was a champion of the dog model for bacterial cell wall containing agents such as liposome encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE). This agent was found to prolong disease free interval and survival in dogs suffering osteosarcoma, and subsequently in children with the same disease, leading to the licensure of this compound in Europe to treat pediatric osteosarcoma (Mifamurtide- Ciba Geigy). A trial with this nonspecific macrophage activator also extended the survival of dogs with splenic hemangiosarcoma (in combination with surgery and doxorubicin/cyclophosphamide), but thus far this agent is not available for veterinary clinical practice. However, a recently licensure was granted for a different Mycobacterial cell wall fraction agent (Immunocidin - Bioniche) which is indicated for use in dogs with mammary tumors. Immunocidin is administered only by intratumoral injection. The entire tumor and a small region of adjacent and underlying tissue are infiltrated using a 20-gauge needle. Injection must be carefully applied to insure full coverage of the tumor bed or treatment may be ineffective. The drug is in an emulsion that must be thoroughly mixed prior to injection, with injection as quickly as possible because the emulsion may begin to separate soon after mixing. The tumor tissue may be very firm and excessive pressure on the syringe plunger may be required to infiltrate the tumor. The injection may produce local pain so use of anesthetics or additional analgesics may necessary. Dosage varies with tumor size but 1 ml should be considered a minimum dose. Treatment may be given once, followed by surgical excision of tumor in 2-4 weeks. The agent is reported to be well tolerated by aged dogs with chronic cardiovascular and renal disease, and may occasionally result in tumor regression in debilitated patients even without surgery for those patients who are poor surgical risks. In this scenario, treatment is repeated every 1-3 weeks, and tumors that fail to respond after 4 treatments should be considered refractory and therapy discontinued. According to the manufacturer s information, eighty-eight percent of dogs treated with immunotherapy only were free of tumor two years later. Individual doses range from 1 to 30 ml (average about 2.5 ml). The average cumulative dose is about 10 ml. Lymphoma immunotherapy Despite great advances with chemotherapy in the treatment of canine malignant

lymphoma, the majority of affected dogs develop chemoresistance and die of this disease. A number of strategies are under consideration to circumvent this resistance that are intended to stimulate antitumor immune responses. These include (but are not limited to) targeting lymphoma antigens with unmodified ("naked") antibodies or antibodies armed with toxins or isotopes, targeting growth factor receptors on lymphoma cells with armed growth factors, lymphoma idiotype vaccines, and whole tumor cell vaccines. An antibody that binds to CD20 on the surface of human B cells (Rituximab ) is now an approved treatment for some forms of human lymphoma. After binding to the target cells, the antibody triggers tumor cell killing through antibody-dependent cell mediated and complement-dependent cytotoxic mechanisms. Unfortunately, this antibody has proven ineffective in binding the target CD20 antigen in veterinary species. However, several companies are engaged in production of canine-specific monoclonal antibodies that may meet this need in the future. A CD19 antigen targeted monoclonal antibody is in trials currently for canine B-cell lymphoma. Targeted immunotherapy The interleukin-2 receptor (IL-2R), normally found on activated lymphocytes, has been identified on the malignant lymphocytes from a number of forms of human lymphoma. In this setting, IL-2R is expressed inappropriately, representing a tumor-associated antigen and not normal expression of an activation molecule. This receptor, composed of three distinct subunits (a, b, g), has been intensively studied as a target for cancer immunotherapy. In human oncology, an antibody against the IL-2Ra subunit has been developed and used either unmodified, armed with a bacterial toxin, or armed with an isotope for the treatment of IL-2R+ human lymphomas. Because this antibody does not recognize IL-2Ra on canine lymphoid cells, an antibody specific for canine IL-2Ra is under development. The majority of canine lymphomas do express IL-2Ra (as well as IL-2Rb and g), suggesting that strategies targeting this receptor in canine lymphoma may have wide applicability. Another approach targeting the IL-2R in lymphoma relies on the ability of this receptor to bind IL-2, the ligand for IL-2R. Thus, an IL-2R-toxin fusion molecule has been licensed for some forms of human lymphoma. The therapy relies on IL-2 to target a toxin directly to IL-2R+ cancer cells. This fusion protein, commercially available as Ontak, is composed of IL-2 bound to diphtheria toxin and it only takes one molecule to kill a tumor cell. Further, because of its unique mechanism of action, chemotherapy resistance is overcome. Several dogs with lymphoma have received Ontak, to our knowledge, without showing conclusive proof of efficacy. The IL-2R status of these dogs' lymphoma cells is unclear and expression of IL-2R is a prerequisite for the rational use of Ontak. More dogs will likely be treated with this molecule in the future although it may be cost prohibitive for many pet owners.