The Future of Canine Cancer Vaccines

Canine Cancer Vaccine Program Shows Early Promise
University of Wisconsin-Madison, January 27, 2006

It wasn't publicized, other than by word of mouth, and still the University of Wisconsin-Madison School of Veterinary Medicine was overwhelmed with requests. Since 1998, the school's oncology department has been producing an anti-cancer vaccine for dogs diagnosed with melanoma. Though it is still an experimental treatment, dog owners from all over the nation have wanted to participate in the study, on the remote chance that this would help their pet.

After promising results from work done in collaboration with cancer specialists from Arizona, California, and Michigan, the school has hired a full-time technician to produce the existing vaccine. The vaccine being used now has undergone a few modifications designed to increase its anti-cancer activity. "Not all dogs with melanoma respond to this treatment," cautions Ilene Kurzman, a researcher in the veterinary medical school's oncology section. "But those that do seem to do quite well."

She would like to continue working on the vaccine in the hope that this innovative anti-cancer strategy will translate into similar novel treatments in people with cancer.

Melanoma, the equivalent of one form of skin cancer in humans, is very aggressive in dogs. It usually manifests itself in or around the mouth or toes. Despite conventional treatment, 75 percent of dogs with oral melanoma will die within one year.

But about 40 percent of dogs with a melanoma tumor present responded to a vaccine created from actual melanoma tumor cells. In about 12.5 percent of the treated dogs, the tumor completely disappeared. While the current results are promising, funding limitations reduce the program's ability to take the next step in improving the vaccine and increasing the percentage of animals that respond, Kurzman says.

According to Kurzman, the vaccine is created from dog melanoma cells that are grown in the laboratory. The cells are treated so they can no longer divide and cause a tumor. DNA is then inserted into these cells, which directs the cells to secrete an immune stimulant. This combination of cells and immune stimulant, when administered as an injection into the patient's skin, has been shown to stimulate the immune system to specifically fight against the melanoma cells.

Dogs that first had surgery for their melanoma and then received vaccine lived cancer-free for approximately twice as long as dogs in previous studies that did not receive the vaccine. Further work is needed to improve the vaccine so that a higher percentage of dogs with melanoma will respond. "It's the closest thing to a miracle I've ever seen," says Maggie Hoefling, of Largo, Florida. Following vaccine therapy, her husband Gus's 14-year-old beagle, Mack, not only lived an additional two years, but thrived. And that's after their local veterinarian gave Mack only four months to live when he was first diagnosed with melanoma. Mack has since died, but he died of congestive heart failure, not cancer, and had gained two more years of quality life.

DNA-Based Vaccine Triples Survival for Dogs with Melanoma
Immune-Boosting Vaccine Also Being Studied in Humans
Memorial Sloan-Kettering Cancer Center

NEW YORK, April 8, 2003 -- The options for treating advanced melanoma are limited -- regardless of whether the patient is a dog or a human. Because this deadly cancer is virtually resistant to chemotherapy and radiation in its late stages, new approaches are being investigated including vaccines that harness the immune system. For nine dogs that naturally developed canine malignant melanoma, treatment with a new DNA-based vaccine more than tripled their median survival from an expected 90 days to an average of 389 days.

The results of this collaboration between the dogs' veterinarians at The Animal Medical Center (AMC) and researchers at Memorial Sloan-Kettering Cancer Center where the DNA-based vaccine had undergone preclinical testing are reported in the April issue of Clinical Cancer Research. The vaccine continues to be studied at AMC. A parallel clinical trial began last fall at MSKCC for people with high risk of melanoma recurrence.

"Most medicines that we use to treat animals are the same as those given to humans," explained Philip J. Bergman DVM, MS, PhD, Head of the Donaldson-Atwood Cancer Clinic and the Flaherty Comparative Oncology Laboratory at The Animal Medical Center and the study's first author. "This vaccine was first tested in the laboratory at MSKCC and then given to dogs with melanoma after receiving approval from the United States Department of Agriculture and the AMC's own Institutional Review Board. We felt it was useful to see if immunotherapy might help these very sick dogs with advanced melanoma since the response rates for standard chemotherapy were extremely poor with no evidence of improved survival."

Canine malignant melanoma (CMM) is the most common oral cancer in dogs and accounts for one out of twenty cancer diagnoses. It is highly aggressive, occurring spontaneously in the mouth, nail bed and foot pad. CMM is most successfully treated in its early stage by surgery. However, the prognosis is not good if there is a late diagnosis or the cancer has spread to another organ. In advanced stages, the median survival is 2 to 3 months.

In this study, nine dogs with advanced melanoma were given four biweekly injections of human tyrosinase DNA vaccine that was constructed at MSKCC's Gene Transfer and Somatic Cell Engineering Facility. The dogs were injected with the vaccine using the Biojector-2000, a needle-less delivery device. They showed no side effects or toxicities with only a mild inflammatory reaction observed at the injection site. Two showed no evidence of disease when they were checked after completion of the vaccine regimen. Four dogs survived for over 400 days with the longest survivor still alive after more than 615 days. The median survival was 389 days.

"Like humans, dogs develop melanoma spontaneously through an interaction of their genes with the environment," said Jedd D. Wolchok, MD, PhD, an oncologist on the Clinical Immunology Service at Memorial Sloan-Kettering and senior author of the study. "By conducting trials in humans and large animals that live in the same surroundings as humans and spontaneously develop cancers, there can be a synergy that we hope will result in improved cancer treatment for all."

The study's co-authors are Josephine McKnight, DVM, Andrew Novosad, DVM, Sarah Charney, DVM, John Farelly, DVM, Ann E. Hohenhaus, DVM, and Diane Craft, BS, of The Animal Medical Center. From Memorial Sloan-Kettering Cancer Center -- Alan N. Houghton, MD, Chief, Clinical Immunology Service; from the Gene Transfer Facility -- Michel Sadelain, MD, PhD, Director; Isabelle Riviere, Ph.D., co- Director; Yusuf Jeffers, and Michelle Wulderk, PhD; from the Laboratory of Tumor Immunology -- Neil Segal, PhD , Polly Gregor, PhD, and Manuel Engelhorn, PhD.

The study was supported by the National Institutes of Health, Swim Across America, Mr. And Mrs. Quentin J. Kennedy Fund, Bioject and Merial Ltd.

Memorial Sloan-Kettering Cancer Center is the world's oldest and largest private institution devoted to prevention, patient care, research, and education in cancer. Our scientists and clinicians generate innovative approaches to better understand, diagnose and treat cancer. Our specialists are leaders in biomedical research and in translating the latest research to advance the standard of cancer care worldwide.

The Animal Medical Center, a not-for-profit veterinary hospital open 24 hours a day every day of the year, specializes in more than 20 areas of medicine and surgery. It is dedicated to providing the highest quality medical care to each one of over 60,000 patient cases seen each year. AMC has served the community in the areas of pet health care, postgraduate education of veterinarians, and clinical investigation of naturally occurring disease in animals.

Cancer Vaccines: A Realistic Therapy or Not?
Rowan Milner, BVSc, Mmed Vet (Med), DECVIM
Small Animal Clinical Services College of Veterinary Medicine, University of Florida

Cancer vaccines differ from conventional vaccines for infectious diseases in as much as they are given after the antigenic insult similar to the old hyperimmune serums. Their main targets are tumor antigens (1). Historically the first vaccines consisted of extracts from pyogenic bacteria or mycobacteria which elicit an immune response (1). An example would be the use of BCG vaccine for equine sarcoid, which when given intra-tumorally stimulated an antitumor immune response in a paracrine fashion. Two recent advances have helped the cause of cancer vaccines and these are; immunological understanding of lymphocyte activation and cytokines; and gene transfer technology (1). The targets of cancer vaccines have been identified in some cancers, mainly melanomas and renal carcinomas. Molecular identification of cancer antigens have identified a number of antigenic sites these are; tissue specific antigens, reactivated embryonic gene products, mutated gene products and viral gene products. The main response to cancers is via the cell mediated immune response. The cells that are responsible for CTL (cytotoxic Tcell lysis) are CD8+ T-lymphocytes (1). In order for there to be a good antitumor effect three important processes must occur; adequate presentation of the cancer antigen must be presented by the MHC-I on the tumor cell or antigen presenting cell; in addition this must occur with costimulation by such complexes as B7-CD28; local elaboration of cytokines (1). Insufficient tumor antigen presentation on MHC-I or lack of costimulation leads to cell anergy, which a mechanism potentially responsible for induction of tolerance to "self" antigens. Other reasons could be lack of recognition of tumor antigen on MHC-I or inadequate CD8+ T-cell activation by "helper" arm (CD4+ T-cells) (1).

Research in the direction of cancer vaccines has followed the following courses (1):

Genetically altered whole-cell tumor vaccines

• Allogenic

• Autologous

• Antigen-Based vaccine strategies

• Vaccination with MHC-1 binding peptides

• Recombinant Viral Vaccines

• Recombinant Bacterial Vaccines

• Nucleic Acid Vaccines

• Dendritic cell vaccines

• Heat shock proteins as carriers of antigen

Historically whole-cell tumor vaccines were used together with adjuvants. These adjuvants often consisted of BCG or Corynebacterium parvum. Although some studies showed moderate clinical responses, these were uncharacterized. The way these vaccines worked appeared to correlate with delayed hypersensitivity reaction to the autologous tumor cells (1). Currently genetically altered whole-cell tumor vaccines seem to be more successful and the response can be characterized. The technique of gene transfer utilizes in most cases a viral vector to implant genes into the cancer cell to enhance the Tcell immune response. Two methods have been studied. The first is genetically altering the cancer cell to produce cytokines (e.g., GM-CSF) which enhance the attraction of antigen presenting cells (APC) such as dendritic cells to the tumor. At the University of Florida we are currently exploring the use of cytokine modified tumor vaccine for cats with vaccine associated soft-tissue sarcomas. The second method is to genetically alter the cancer cell to become a professional APC, expressing the ability to present tumor antigen on major histocompatibility complexes (MHC). These techniques use both ex-vivo and in-vivo methods. A number of technical problems exist, in broad terms these are; limiting viral induced genes or gene products to the tumor (2); and switching off production of the induced cytokine. Because growing cancer cells ex-vivo can be difficult, bystander cells such as human vero cell can be utilized. These cells are histoincompatible and therefore for example, the cats own immune system would ultimately destroy the cells thus switching off the cytokine production. This technique has been employed in the veterinary field to treat vaccine associated sarcoma in cats (3). Results using this technique were promising. Limitations of autologous whole-cell tumor vaccines relates to the expansion of tumor cells from individuals, which can be technically difficult, in addition whole cells constitute an inefficient source of antigen which is required for tumor rejection (1). Currently, allogenic whole-cell tumor vaccines maybe more effective, they are prepared ex-vivo from existing tumor cell lines. Research has shown that the technique is effective (1).

Another approach is antigen-based vaccines. These strategies have three main requirements to be successful designs, these are; identification of common antigens recognized by T-cells and expressed by the majority of cancer patients; identification of a single antigen that can serve as a tumor rejection target in-vivo; development of recombinant vaccine strategies that can generate antigen specific immunity (1). Identification of common antigens has been successful, mainly for cancers such as melanoma. The techniques used to identify common antigens include genetic and biochemical approaches (1).

As mentioned previously tumor antigens fall into four main categories these are:

• Tissue specific antigens, for example these are commonly shared antigens among malignant melanoma cancers.

• Reactivated embryonic gene products. Mage-1 is a common product found in melanomas. These products must be specifically recognizable by T cells.

• Mutated gene products. These can be oncogene or tumor suppressor gene products e.g., p53.

• Viral gene products. Examples of these would be Burkitt's lymphoma and Epstein - Barr virus, possibly FeLV could also be targeted in feline lymphoma.

Since most work has been done on melanomas it is hoped that other tumors will be similar and have common antigens. Two approaches have evolved in the development of antigen-based tumor vaccines, these can be divided into DNA-based vaccines that deliver the gene encoding the antigen and peptide- or protein-based vaccines that deliver antigens pulsed on to APCs or mixed with adjuvants (1). An example of DNA-based vaccine include the injections of "naked" or plasmid DNA intramuscularly using needle-free jet delivery device. Currently a phase one clinical research trial in dogs injected with DNA plasmid encoding for a mouse melanoma antigen was found to be effective with durable remission of metastatic disease in one dog (4). Vaccines employing APCs pulsed with tumor associated antigen have also been successful (5). Once again melanomas in dogs were used. Recombinant viruses and bacterial vaccines have also been developed. Utilizing the natural cytotoxic effect that viruses have which attracts APCs is one mechanism. Viruses would have to be restricted to the tumor for this to be effective. It has been known for number years that spontaneous remission have occurred in people that have had viral infections or vaccination. In addition modified viruses with introduced genes can target bone-marrow derived dendritic cells causing them to express tumor associated antigen or encoded costimulatory molecules and enhance the tumor killing effect. Recombinant bacterial vaccines involve the use of genetically engineered bacteria. Bacterial strains of Salmonella, BCG and Listeria monocytogenes have two characteristic which are beneficial. They posses the ability to infect the host via the enteric route, thus providing for oral vaccine use. Secondly, recombinant L. monocytogenes has a two-phase intracellular life cycle. The bacterium infects monocytes and macrophages and occupies phagolysosomes. The bacterium secretes Listeria lysin O which destabilizes the lysosome and allows the bacteria to enter the cytoplasm. Based on the known ways that APCs present antigen either via MHC-1 (cytosolic phase) or MHC-2 (phagolysosome phase) the bacteria can be manipulated to present tumor specific antigen either via MHC-1 to CD-8 T-lymphocytes or via MHC-2 to CD-4 T lymphocytes.

Dendritic cell (DC) vaccines have recently received attention as cancer vaccine because of their ability to stimulate antigen specific T-cells (1;6). Dendritic cells are 100 to 1000 more potent than macrophages in stimulating a response in T-cells, this is due to their higher expression of MHC, cytokine and costimulatory receptors. Based on these characteristic DC, may serve as good presenters of tumour specific antigen to CTL. Currently a number of studies have investigated loading DC with autologous cancer antigen ex-vivo and vaccinating the patient. A newer technique is to use fusion proteins, which are combination of cancer cells and DC.

Heat shock proteins (HSP) can also be used as carriers of antigen (1). HSP are proteins produced by genes that are induced when intracellular conditions promote denaturing of proteins e.g., heat. They act to protect other proteins and aid in the refolding of denatured proteins. HSP can serve as natural biological adjuvants; they also have the capacity to bind a wide array of proteins. The HSP that are an extract from a specific tumour e.g., HSP70 are unique to that tumor and can stimulate an immune response (1). In conclusion cancer vaccine will in the near future become more of a reality as more tumor antigens are identified. Results from research in experimental animals and human and animal clinical trials are promising, however these vaccines are likely to be used as an adjunct to therapies currently used in oncology.

Definitions

• Autologous--same animal

• Allogeneic--same species

• Xenogeneic--different species.

References
1.  Jaffee EM, Pardoll DM. Cancer-Specific Vaccines. In: Mendelsohn J, Howley PM, Israel MA, Liotta LA, editors. The Molecular Basis of Cancer. Philadelphia: W.B. Saunders Company, 2001: 573-588.
2.  Weld KJ, Mayher BE, Allay JA, Cockroft JL, Reed CP, Randolph MM et al. Transrectal gene therapy of the prostate in the canine model. Cancer Gene Ther 2002; 9(2):189-96.
3.  Quintin-Colonna F, Devauchelle P, Fradelizi D, Mourot B, Faure T, Kourilsky P et al. Gene therapy of spontaneous canine melanoma and feline fibrosarcoma by intratumoral administration of histoincompatible cells expressing human interleukin-2. Gene Ther 1996; 3(12):1104-12.
4.  Wolchok JD, Houghton AN, Bergman PJ. Of Mice and Men (and Dogs): Xenogeneic DNA Vaccines for Melanoma. 2002. Ref Type: Personal Communication
5.  Rodriguez-Lecompte JC, Gyorffy S, Majumdar A, Gauldie J, Wan Y. Dendritic cells transduced with adenovirus expressing human TERT or gp100 tumor-associated antigens as cancer vaccines. 2002. Ref Type: Personal Communication
6.  Onaitis M, Kalady MF, Pruitt S, Tyler DS. Dendritic cell gene therapy. Surg Oncol Clin N Am 2002; 11(3):645-60.

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