Of mice and men (and dogs!) – vaccines for canine malignant melanoma (Proceedings)

Canine malignant melanoma (CMM) of the oral cavity, nail bed, foot pad and mucocutaneous junction is a spontaneously occurring, highly aggressive and frequently metastatic neoplasm. CMM is a relatively common diagnosis representing ~ 4% of all canine tumors and it is the most common oral tumor in the dog. CMM and advanced human melanoma (HM) are diseases that are initially treated with aggressive local therapies including surgery and/or fractionated radiation therapy; however, systemic metastatic disease is a common sequela. Based on these similarities, CMM appears to be a good clinical model for evaluating new treatments for advanced HM.

Canine patients with advanced disease (WHO stage II, III or IV) have a reported median survival time of 1-5 months with standardized therapies. A combination of hypo-fractionated radiation therapy and chemotherapy have a reported median survival time of one year in stage I oral CMM. Human patients with deep AJCC stage II or stage III disease (locally advanced or regional lymph node involvement) have at least a 50% chance of recurrence after surgical resection; patients with stage IV melanoma (distant metastases) have a median survival of less than ten months and most of these patients eventually die of melanoma. Standard systemic therapy is dacarbazine chemotherapy in HM, and carboplatin chemotherapy in CMM. Unfortunately, response rates to chemotherapy in humans or dogs with advanced melanoma range from 8-28% with little evidence that treatment improves survival. It is easily evident that new approaches to this disease are desperately needed and multiple methodologies have been reported to date.

Active immunotherapy in the form of vaccines represents one potential therapeutic strategy for melanoma. The advent of DNA vaccination circumvents some of the previously encountered hurdles in vaccine development. DNA is relatively inexpensive and simple to purify in large quantity. The antigen of interest is cloned into a bacterial expression plasmid with a constitutively active promoter. The plasmid is introduced into the skin or muscle with an intradermal or intramuscular injection. Once in the skin or muscle, professional antigen presenting cells, particularly dendritic cells, are able to present the transcribed and translated antigen in the proper context of major histocompatibility complex and costimulatory molecules. The bacterial and plasmid DNA itself contains immunostimulatory sequences that may act as a potent immunological adjuvant in the immune response. In clinical trials for infectious disease, DNA immunization has been shown to be safe and effective in inducing immune responses to malaria and human immunodeficiency virus. Although DNA vaccines have induced immune responses to viral proteins, vaccinating against tissue specific self-proteins on cancer cells is clearly a more difficult problem. One way to induce immunity against a tissue specific differentiation antigen on cancer cells is to vaccinate with xenogeneic antigen or DNA that is homologous to the cancer antigen. It has been shown that vaccination of mice with DNA encoding cancer differentiation antigens is ineffective when self-DNA is used, but tumor immunity can be induced by orthologous DNA from another species.

We have chosen to target defined melanoma differentiation antigens of the tyrosinase family. Tyrosinase is a melanosomal glycoprotein, essential in melanin synthesis. The full length human tyrosinase gene was shown to consist of five exons and was localized to chromosome 11q14-q21. Immunization with xenogeneic human DNA encoding tyrosinase family proteins induced antibodies and cytotoxic T cells against syngeneic ±6 melanoma cells in C57BL/6 mice, but immunization with mouse tyrosinase-related DNA did not induce detectable immunity. In particular, xenogeneic DNA vaccination induced tumor protection from syngeneic melanoma challenge and autoimmune hypopigmentation. Thus, xenogeneic DNA vaccination could break tolerance against a self tumor differentiation antigen, inducing antibody, T-cell and anti-tumor responses.

Experimental Design

From April 2000 to present, approximately 500 dogs with previously histologically confirmed spontaneous malignant melanoma were treated with xenogeneic DNA vaccinations. Pre-trial evaluation included complete physical examination, a complete blood count and platelet count, serum chemistry profile, urinalysis, lactate dehydrogenase, anti-nuclear antibody, and three-dimensional measurements of the primary tumor if present (or maximal tumor size from medical records if patient was treated prior to pre-trial considerations). For evaluation of metastatic disease, 3-view radiographs of the thorax were obtained and regional lymph nodes were evaluated with fine needle aspiration/cytology and/or biopsy/histopathology. All dogs were clinically staged according to the WHO staging system of stage I (tumor < 2 cm diameter), II (tumors 2-4 cm. diameter, negative nodes), stage III (tumor > 4 cm. and/or positive nodes) or stage IV (distant metastatic disease). The numbers of previous treatments with surgery, radiation and/or chemotherapy were recorded. Dogs with WHO stage II, III or IV histologically confirmed malignant melanoma were allowed entrance onto the study due to the lack of effective available systemic treatments. Due to a strong safety profile, dogs with stage I melanoma were allowed inclusion from 2005 on. Additional entry criteria included: an estimated life expectancy of 6 weeks or more, free of clinically detectable brain metastases, no previous therapy (surgery, radiation and/or chemotherapy) for at least 3 weeks and no serious intercurrent medical illnesses. Written consent for entry onto this trial was obtained from each dog's owner prior to entry onto the study; this consent included request for necropsy upon death due to any reason. These studies were performed under Animal Medical Center IRB approval.

Cohorts of three dogs each received increasing doses of xenogeneic plasmid DNA encoding either human tyrosinase (huTyr; 100, 500 or 1500mcg), murine GP75 (muGP75; 100, 500 or 1500mcg), murine tyrosinase (muTyr; 5 dogs at 100mcg or 500mcg each), or muTyr ± HuGM-CSF (9 dogs at 50mcg muTyr, 3 dogs each at 100, 400 or 800mcg HuGM-CSF, or 3 dogs each at 50mcg muTyr with 100, 400 or 800mcg HuGM-CSF) intramuscularly biweekly for a total of 4 vaccinations in the left caudal thigh with the Biojector 2000 jet delivery device with #3 (intramuscular) Bioject syringes. The Biojector2000 is a carbon dioxide powered jet delivery device which is FDA approved for administration of intramuscular injections and has been used in DNA vaccine clinical investigations. At present, dogs are receiving 50mcg MuTyr as outlined above except with the Vitajet/PetJet spring-loaded needle-free injection device. Subjective pain level responses and post-vaccinal presence of a wheal or other reaction were assessed and recorded by the veterinarian administering the DNA vaccination. The dogs did not receive any concomitant treatments during the course of vaccination.

Results

The signalments of dogs on this study have been similar to those in previously reported CMM studies. No toxicity was seen in any dogs receiving the aforementioned vaccines with the exception of minimal to mild pain responses at vaccination, one muGP75 dog experienced mild aural depigmentation, and one muTyr dog has experienced moderate foot pad vitiligo. Dogs with stage II-III loco-regionally controlled CMM across the xenogeneic vaccine studies have a Kaplan-Meier (KM) median survival time (MST) of > 2 years (median not yet reached). The KM MST for all stage II-IV dogs treated with huTyr, muGP75 and muTyr are 389, 153 and 224 days, respectively. The KM MST for stage II-IV dogs treated with 50mcg MuTyr, 100/400/800mcg HuGM-CSF or combination MuTyr/HuGM-CSF are 242, 148 and > 900 (median not reached, 6/9 dogs still alive) days, respectively. For dogs on the Phase Ib MuTyr/HuGM-CSF/Combination trial, significant differences in MST were noted across pre-vaccination stage (stage IV MST = 99 days, stage III = 553 days and stage II > 401 days, P < .001). The results from dogs vaccinated with huTyr were published in 2003 (Bergman et al, Clin Cancer Res 2003).

Development Of Specific Anti-Tyrosinase Humoral Immune Responses

We have recently investigated the humoral responses of dogs receiving HuTyr as a potential explanation for the long-term survivals seen in some of the dogs on this study. Utilizing standard ELISA with mammalian expressed purified human tyrosinase protein as the target of interest (kind gift of C Andreoni & JC Audonnet, Merial, Inc.), we have found 3/9 dogs with 2-5 fold post-vaccinal humoral responses compared to pre-immune sera. We have confirmed these findings utilizing a flow-cytometric-based assay of pre- and post-vaccinal sera in permeabilized human SK-MEL melanoma cells expressing endogenous human tyrosinase. Interestingly, the three dogs with post-vaccinal anti-HuTyr humoral responses are dogs with unexpected long-term tumor control (Liao et al, Cancer Immunity, 2006). Additional studies are ongoing investigating the humoral responses from dogs on the other xenogeneic DNA vaccines as well as T-cell based assays utilizing ELISPOT and intracellular cytokine staining assays.

Conclusions

The results of these trials demonstrate that xenogeneic DNA vaccination in CMM is: 1) safe, 2) develops specific anti-tyrosinase humoral immune responses, 3) potentially therapeutic with particularly exciting results in stage II/III local-regional controlled disease and dogs receiving MuTyr/HuGM-CSF combination, and 4) an attractive candidate for further evaluation in an adjuvant, minimal residual disease Phase II setting for CMM. A safety and efficacy USDA licensure multi-institutional trial investigating HuTyr in dogs with locally controlled stage II/III oral melanoma was initiated in April, 2006. Human trials of xenogeneic tyrosinase DNA vaccination have recently initiated. In late March 2007, we received conditional licensure from the USDA for the canine melanoma vaccine. This represents the first US-government approved vaccine for the treatment of cancer.

In summary, CMM is a more clinically faithful therapeutic model for HM when compared to more traditional mouse systems as both human and canine diseases are chemoresistant, radioresistant, share similar metastatic phenotypes/site selectivity, and occur spontaneously in an outbred, immuno-competent scenario. In addition, this work also shows that veterinary cancer centers and human cancer centers can work productively together to benefit veterinary and human patients afflicted with cancer.

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