Autologous Cancer Vaccines: A Precision Immunotherapy Strategy for Veterinary Cancer Patients

Tumor Antigens

Somatic mutation of tumor cells results in extreme antigenic heterogeneity between tumors (4). Broadly, these antigens can be placed into two categories, tumor-associated antigens (TAA) and tumor-specific antigens (TSA). TAA are comprised of normal “self” proteins that, because of central immune tolerance, may not be sufficiently immunogenic to create an effective immune response (5). Examples of TAA studied in veterinary oncology include tyrosinase, present in canine melanoma, and HER2/neu, found in canine osteosarcoma (6, 7). TSA are comprised of mutated, novel, proteins that have no central tolerance and are often highly immunogenic (5). These so-called neoantigens (NeoAg) are attractive immunotherapy targets because of their cancer specificity and propensity for immune recognition (8).

Another source of antigens is the tumor stroma, which is a complex combination of cells and extracellular matrix that support cancer growth and progression. The stroma actively supports the cancer structurally and elaborates growth signals to the cancer cells. Further, the stroma contributes to local immunosuppression by inhibiting the immune response and restricting T cell infiltration into the tumor (9). It stands to reason that an immune response directed against both cancer cells and the stroma represents an enhanced therapeutic possibility. Additionally, tumor stromal cells are genetically and antigenically more stable compared to tumor cells with high rates of somatic mutation; thus, an immune response that includes stromal targets offers a better chance for long-term tumor control (4).

Therapeutic Cancer Vaccines

The goal of therapeutic cancer vaccines is to expand a population of effector T cells capable of eliminating cancer. As noted above there are myriad antigens potentially useful for creating cancer vaccines. Vaccines created with TAA have been studied in dogs. One example is the Canine Melanoma Vaccine, DNA (Oncept^®; Boehringer Ingelheim, Duluth, GA, USA). This vaccine contains a DNA plasmid that encodes human recombinant tyrosinase, and this xenogeneic approach helps overcome the dog’s central immune tolerance to native tyrosine (10). Oncept^® was reported to be well tolerated by dogs to prolong the survival of dogs with advanced oral melanoma and adequate locoregional disease control (11). A recent review notes the contradictory outcome data published in the veterinary literature since 2011, making the survival benefit for dogs less clear (12). Because tyrosinase is only found in melanoma, Oncept^® is limited to a single cancer type; however, it has been used in cats and horses (13-15).

Another example of a TAA-targeting vaccine is the canine osteosarcoma vaccine, that utilized an attenuated Listeria monocytogenes expressing a chimeric human HER2/neu fusion protein. This vaccine was shown to break tolerance in over 80% of dogs with HER2/neu⁺ osteosarcoma treated with the vaccine after amputation and chemotherapy, and improved the 1-, 2-, and 3-year survival rates compared to the control group treated with amputation and chemotherapy alone (7). In a safety study including 49 dogs with osteosarcoma, 60% of administered doses were associated with an adverse event, and the live Listeria vector was associated with positive Listeria cultures in 8% of treated dogs, a noteworthy finding given the zoonotic potential of the organism (16). Although the canine osteosarcoma vaccine was granted a conditional license by the USDA in 2017 (17), it is no longer under commercial development.

With tumor specificity and lack of peripheral and central immune tolerance, TSA, specifically NeoAg, are an attractive target for cancer vaccines (18, 19). NeoAg based cancer vaccines may increase therapeutic efficacy and lower the probability of autoimmune-type adverse events, compared to tumor-associated antigens (20, 21). In murine models, NeoAg vaccines have demonstrated ability to induce intratumoral expansion of NeoAg-specific cytotoxic CD8⁺ T cells as well as inducing proliferating and stem-like NeoAg-specific CD8⁺ T cells (22-24). This expansion of NeoAg-specific cytotoxic CD8⁺ T cells results in tumor reduction and increased overall survival with limited toxicity. The first human clinical trial of NeoAg vaccines launched in 1997; since then, more than 100 clinical trials have been initiated (25). In one such clinical trial, vaccination of acute myeloid leukemia patients resulted in a 15.7-fold expansion of CD8⁺ cancer-specific T cells. Moreover, after 57-months, 12 of 17 patients were still alive, disease-free, having only experienced transient grade 1 and 2 adverse events (26).

Identification of candidate NeoAg peptides requires sequencing of the tumor genome to identify expressed protein-coding sequences against a normal background, followed by MHC-I predictive algorithms, and in vitro validation assays (8). Despite best efforts, the prediction of immunologically meaningful NeoAg peptide candidates remains elusive (27, 28). To date, two predictive strategies have been described in veterinary medicine. The first is for canine melanoma vaccines, which models strong binding affinity to the dog leukocyte antigen DLA 88*002:01, along with modified human MHC-1 typing and binding affinity software tools to predict potentially immunogenic NeoAg (29). The second is for the development of frame shift peptide vaccines for canine cancer based on shared NeoAg, which can be predicted by utilizing custom multiplex arrays (30). Frame shift peptides represent an exceedingly small, highly immunogenic subset of potential NeoAg within a tumor. Because the majority of NeoAg are unique to individual patients (5), and a resource-intensive methodology is required to identify candidate NeoAg, this approach may be best suited to developing a veterinary cancer vaccine for a single cancer type, rather than creating a bespoke vaccine for an individual.

With the limitations of cancer vaccines targeting TAA, and the current technical challenges in predicting immunogenic NeoAg in veterinary species, a compelling argument can be made for the idea of using autologous cells and tissue as an antigen source. Autologous cancer vaccines (ACV) are derived from the patient’s own tumor tissue, allowing their immune system to be exposed to a wide array of antigens, including TAA, TSA and NeoAg, and, in the case of whole tissue vaccines, stromal antigens. Although one of the earliest forms of cancer immunotherapy studied, ACVs represent an elegant solution for the antigen selection problem created by tumor heterogeneity by presenting the immune system with antigens directly applicable to a specific patient’s tumor. Further, ACVs are agnostic to both tumor type and host species, making this an attractive immunotherapy option for veterinary cancer patients.

Development of Autologous Cancer Vaccines

The early history of cancer immunotherapy in veterinary medicine, particularly the development of ACV, is fascinating; however, a complete review is beyond the scope of this work. Decades before the recognition of T cells, or the discovery of dendritic cells and natural killer cells, Suguira and Benedict demonstrated that inoculation of chickens with heat-inactivated Rous sarcoma tissue resulted in marked resistance to subsequent growth of viable tumor tissue injected into naïve birds (31, 32). Further work revealed that inactivation by heat, chemicals, or drying of tissue from a transplantable chicken lymphoid tumor could be used to effectively immunize naïve chickens against challenge with injected live tumor tissue (33). Investigations using rats demonstrated that regression of methylcholanthrene-induced sarcomas via restriction of blood supply rendered the animals resistant to tumor growth following inoculation with tumor cells, and immunity was less likely to occur with rapidly growing tumors (34-36). Together, these findings formed the theoretical basis for tumor immunotherapy using autologous cancer cells and tissue. Subsequent work in rabbits with Shope papillomas showed that groups given an ACV had tumor regression rates up to 90%, compared to 25% in the untreated controls, and tissue handling and storage affected vaccine efficacy (37). These observations laid the groundwork for ensuing decades of ACV research in veterinary oncology.

ACV are usually prepared as inactivated whole, or lysed, tumor cells or tissue, and some formulations include the supporting tumor matrix and associated antigens. A common approach to production of whole tumor cell ACV is to enzymatically digest the tumor tissue, which may destroy some tumor antigens (38, 39), and then subject the tumor cells to lethal UV irradiation (40, 41). Mechanical cell dissociation and chemical inactivation has also been described as a method to produce whole tissue ACV (42). ACV prepared from cancer cells separated from the tumor matrix usually undergo steps which require in vitro growth and expansion of the cells. Cell culture is expected to differentially select for some cell types over others and alter the antigenic profile vis-à-vis native cancer cells (43, 44). Tumor cell lysates used as ACV are commonly produced by either ultraviolet B ray irradiation or repeat cycles of freezing and thawing (4).

Various strategies to improve the immune response to ACV in veterinary oncology have been described. The most common method is including an adjuvant in the vaccine formulation. Early work utilized bacterial adjuvants, including Vibrio Cholera neuraminidase (VCN) in dogs with mammary cancer (45), Freund’s complete adjuvant in dogs with lymphoma (46), and Bacillus Calmette–Guérin (BCG) in cats with fibrosarcoma (47). More recently, streptavidin was evaluated as an adjuvant for ACV in dogs with a variety of cancers (48). Unmethylated CpG motifs, frequent in bacterial DNA and a ligand for Toll-like receptor-9 (49), have been used as ACV adjuvants in a variety of canine cancers (40, 50). A bioactive extracellular matrix adjuvant (SIS), that attracts macrophages to the injection site, allows maturation of antigen presenting cells, and upregulates cytokines that activate adaptive immunity (51), has been used in ACV used to treat a variety of cancers in dogs, cats, and horses (42, 52-54).

Another method to improve the immune response to ACV includes plasmid transfection of cancer cells to induce expression of human granulocyte-monocyte colony stimulating factor (GM-CSF) (55-57), human interleukin-2 (IL-2) (58), or Emm55, a serotyping protein of Streptococcus pyogenes (59, 60). Transfected ACV have been administered to dogs and horses. In general, clinical evaluations of these approaches have been limited by either the small number of animals included and/or by lack of objective antitumor responses. An exception were results that demonstrated significant increases in the proportion of animals that were local disease-free and metastasis-free versus controls. That particular approach combined suicide gene therapy with a subcutaneous vaccine composed by formolized tumor cells and irradiated xenogeneic cells producing human interleukin-2 and granulocyte–macrophage colony-stimulating factor (58).

Interest in driving functional tumor-specific T cell responses led to the exploration of dendritic cell (DC) vaccines. This involves harvesting DCs from the patient via leukapheresis, exposing them to tumor cell lysate from the patient’s cancer, then infusing the activated DCs into the patient. DC ACV have been studied in dogs using tumor cell lysates from squamous cell carcinoma, melanoma, histiocytic sarcoma, and hemangiosarcoma, and in horses with sarcoid and squamous cell carcinoma (61-66). A similar approach, using autologous CD40-activated B cells loaded with tumor RNA, was evaluated in dogs with lymphoma (67, 68). These studies have primarily demonstrated induction of immunologic responses suggestive of anti-tumor activity, and some limited clinical data supports further investigation with greater numbers of animals, particularly with respect to canine hemangiosarcoma and non-Hodgkin’s lymphoma (66, 68).

A novel approach to cancer immunotherapy uses autologous tumor mRNA that is packaged into lipid particle aggregates as a way to enhance payload packaging and immunogenicity (69). Initial trials with canine glioma patients found that median overall survival improved from 35 days (based on historical controls) to 139 days in animals receiving the experimental treatment. Though these results are based on 5 dogs per treatment group, the results are encouraging and merit further investigation.

Regardless of specific methodology, ex vivo manipulation and inactivation of tumor cells, plus a component to enhance the immune response, results in an ACV, that when administered to the patient, stimulates humoral and cell-mediated immune responses against multiple cancer-specific antigens (5). The variation in formulations of ACV studied in veterinary medicine over time reflects the evolution of both the understanding of the immune system and available technology, contemporaneously with the prevailing interest of the scientific community that influences research funding.

Veterinary Applications of Autologous Cancer Vaccines

With 50 years of clinical experience using ACV in veterinary cancer patients, there are a multitude of peer-reviewed publications. Common to the veterinary literature, these studies are often small, may have a retrospective study design, and inconsistently report adverse events and clinical outcomes. Other studies were done in companion animals as a prelude to clinical investigation in humans. Given the wide array of ACV formulations described above, it creates challenges in understanding the relative benefits between different ACV. Regardless of the limitation of individual studies, when viewed in toto, they demonstrate the relative safety and potential efficacy of ACV in veterinary cancer patients. Although a comprehensive review is beyond the scope of this paper, there are several noteworthy studies presented herein that highlight the potential for ACV in veterinary oncology. Examples of clinical studies to evaluate ACV preparations in canine patients are shown in Table I.

Given the frequency of canine mammary gland tumors, and the budding interest in comparative oncology, it is not surprising the first clinical report in the veterinary literature described the use of an ACV comprised of mitomycin treated cancer cells adjuvanted with VCN in dogs with mammary cancer (45). Dogs with >1 mammary mass were randomly assigned to treatment groups without knowledge of tumor type. All but one of the masses was surgically removed to create the ACV and to allow for histologic diagnosis. Treatment consisted of 2 subcutaneous doses of ACV given on the day of surgery. The group receiving an ACV consisting of only mitomycin treated cells (n=12) had partial response in 2 dogs (approximately 65% decrease in tumor volume), stable disease in 2 dogs, and progressive disease in 8 dogs. The group given an ACV with mitomycin treated cells and VCN (n=15) had partial response in 10 dogs (observed decreases in tumor volumes ranging from 78 to 96%, for up to 300 days), stable disease in 2 dogs, and progressive disease in one dog. Although a variety of mammary cancers were included in this study, and groups were not balanced with respect to tumor type, this study demonstrates the potential for ACV to be a primary cancer treatment, as well as the importance of the adjuvant in creating a meaningful clinical response.

In a subsequent study, 79 dogs with >1 palpable mammary mass, were randomly assigned to treatment groups, then underwent surgical removal of all but one mammary mass as described above. Dogs were treated with ACV containing mitomycin treated cancer cells and adjuvanted with variable amounts of VCN, and containing different cells counts according to assigned treatment groups (70). From this work, it was discovered that dogs administered 2×10⁷ tumor cells subcutaneously had the most consistent regression of their remaining tumor mass, whereas dogs administered 2×10⁶ tumor cells had transient regression followed by tumor progression. Dogs receiving repeated doses of 2×10⁶ tumor cells every 4 weeks for three doses also had transient tumor regression followed by progression. All dogs treated with 1×108 tumor cells had tumor progression, and most developed early metastasis. Those dogs that were given their ACV doses as multiple intradermal injections at different sites had similar positive responses as dogs given 2×10⁷ tumor cells subcutaneously, even when receiving more than 1×10⁸ tumor cells. Additionally, dogs were skin tested for delayed type hypersensitivity (DTH), which varied based on both cell count administered and VCN concentration used in the ACV. These findings confirm the ability of this ACV formulation to cause regression of mammary masses in the gross disease setting. The positive DTH response observed demonstrated a cell-mediated immune response to the vaccine antigens.

Other examples of objective tumor responses from ACV as a primary cancer treatment include: the regression of experimentally-induced Feline Sarcoma Virus (FeSV) sarcomas in cats treated with a BCG adjuvanted ACV (47); melanoma regression in 92% of horses treated with an irradiated ACV (71); complete resolution of all cutaneous papilloma lesions in 9 dogs treated with a heated tumor lysate ACV (72) or in 11 dogs treated with an ACV comprised of autoclaved tumor cells and non-pathogenic Bacillus sp. (73); and a complete response in 4 out of 5 dogs with malignant carcinoma (3 mammary gland, 1 rectal) treated with a DC ACV injected intratumorally with concomitant IFN- γ (74). Interpretation of results from these studies is limited by the relatively small numbers of animals used and by inconsistent anatomical tumor location and histological character.

There is also a role for ACV use in the adjuvant setting following treatment of the primary tumor. In an open-label controlled study of canine malignant melanoma, 493 dogs were enrolled and, based on owners’ wishes, assigned to either the surgery only control group (ST), or the treatment group (CT) (75). Dogs in the CT group received a local injection of lipoplexes containing suicide genes and the gene for canine interferon-β into the surgery site, as well as the ACV. The ACV was comprised of processed tumor cells mixed with lipoplexes containing genes for human IL-2 and GM-CSF. Beginning at the time of surgery, the ACV was administered as a subcutaneous injection, once per week for 5 weeks, then every other week for 5 doses, monthly for 5 doses, every 3 months for 5 doses, then every 6 months until relapse or death. Based on the pathological interpretation of the surgical margins, dogs were stratified in to complete and incomplete resection. Most of the enrolled dogs had oral melanoma. When considering only those dogs that had complete surgical excision (n=249), the overall median survival time of 704 days in the CT group (n=185), was significantly longer than 101 days in the ST group (n=64; p<0.00001). The median melanoma-specific survival was >2,251 days in the CT group, compared to 109 days in the ST group (p<0.00001). Over the course of the study, 118 dogs in the CT group died, and nearly 60% of those deaths were due to causes unrelated to melanoma. Moreover, the 6-year survival rate for the CT group was 29%.

ACV may also be useful in the adjuvant setting for patients with established metastasis. A recent retrospective study describes the use of an ACV in a small number of dogs with metastatic hemangiosarcoma following splenectomy (52). The ACV was comprised of whole tumor tissue that was mechanically dissociated, chemically inactivated, and combined with SIS, bioactive extracellular matrix adjuvant. The ACV was administered as a subcutaneous injection once weekly for 3 doses. Outcomes were compared to historical data. Dogs with metastatic hemangiosarcoma treated with the ACV (n=8) had a significantly longer median survival time (142 days), compared to 42 dogs treated with surgery alone (41 days; p<0.001). Survival time of the ACV group was similar to the survival time of 23 dogs treated with maximum tolerated dose chemotherapy. There were no adverse events noted in the ACV group. This study also explored the mechanism of action of the ACV and demonstrated that the ACV was able to stimulate the upregulation of MHC-II and CD80 in cultured canine macrophages. These signals are markers of antigen presentation and portend T cell activation ability in vivo. Although this was a small, retrospective study, it demonstrates the potential for ACV in patients with disseminated disease, and it provides some additional insights into how these vaccines interact with antigen presenting cells.

The role of adjuvant ACV in veterinary oncology is highlighted by the recent announcement in March 2025 that the U.S. Department of Agriculture – Center for Veterinary Biologics (USDA-CVB) has accepted pivotal efficacy and safety data from a canine osteosarcoma study of ELIAS Cancer Immunotherapy (ECI^®, ELIAS Animal Health, Olathe, KS, USA) and has issued full approval for an Autologous Prescription Product license for the treatment of bone cancer in dogs (76). The essential component of ECI^® is an ACV used to activate cancer antigen-specific T cells which are later harvested by leukapheresis, expanded ex vivo, and infused into the patient. In a pilot study of ECI^®, dogs with histologically confirmed appendicular sarcoma underwent amputation, and of the 14 dogs enrolled, 10 dogs successfully completed the full protocol of ACV, leukapheresis, and T cell infusion with concurrent human recombinant IL-2 administration (77). The median overall survival for all dogs (intent-to-treat basis) was 415 days, with a 36% 2-year survival rate; this is notable given modest survival expectations in dogs treated with amputation alone (median survival time of 134 days, 2% 2-year survival rate) (78). Despite the small sample size, these results demonstrate the potential for autologous therapies in managing aggressive cancers in animals.

Lymphoma is a commonly encountered cancer that has been treated with a variety of ACV formulations. Because lymphoma can be a rapidly fatal disease of dogs, ACV protocols are typically combined with chemotherapy in what is known as chemoimmunotherapy. In the earliest reported work on canine lymphoma, dogs with multicentric lymphoma were randomized to treatment groups, then underwent surgical removal of a lymph node for histopathologic confirmation and production of a chemically modified ACV adjuvanted with Freund’s complete adjuvant (79). After surgery, dogs underwent an 8-week combination chemotherapy protocol comprised of vincristine, cytosine arabinoside, cyclophosphamide, L-asparaginase, and prednisone. All dogs were in complete remission before starting the ACV protocol, which was administered by intramuscular injection on weeks 10, 11, 12, 14, and 16 after initiation of chemotherapy. Dogs in the control group received placebo injections of vitamin B12 on the same schedule. At the time that clinical relapse was noted, the dogs repeated the cycle of chemotherapy or chemoimmunotherapy, dependent on their assigned treatment. The mean survival of the chemoimmunotherapy dogs (n=11) was 348 days compared to 197 days for the chemotherapy only group (n=9). Notably, 5 of the chemoimmunotherapy dogs survived well beyond a year (ranging from 435 to >568 days). Remarkably, the dog that was still alive at day 568 had only gone through the initial round of chemoimmunotherapy. Additional details and longer follow up on this cohort of dogs were published and revealed the chemoimmunotherapy dogs had 1- and 2-year survival rates of 45.5% and 18.2%, respectively, compared to 0% 1-year survival in the chemotherapy only group (80). At the time the follow up results were published, two dogs in the chemoimmunotherapy group were still alive; a 5-year-old male St. Bernard, stage IVa at presentation, was still alive at 933 days after a single round of chemoimmunotherapy, and a 13-year old female mixed breed dog, stage IIIa at presentation, was still alive at 780 days after 2 rounds of chemoimmunotherapy, with the second round starting at day 208. Although adverse events were not specifically reported, there was a mention of some dogs developing sterile abscesses at the site of ACV injection, which was attributed to the oil emulsion component of the adjuvant. The long-term survival observed in the dogs with multicentric lymphoma treated with chemoimmunotherapy was remarkable given the suboptimal chemotherapy component.

The interest in chemoimmunotherapy for canine lymphoma has continued. Results from a randomized placebo control double-blind clinical trial in dogs with diffuse large B cell lymphoma have been reported. Dogs were randomized to receive either combination chemotherapy with an ACV (n=12) created from the dog’s homogenized tumor tissue processed with hydroxylapatite (HA), to assist in purifying heat shock proteins (HSP), and suspended in carboxymethylcellulose (CMC), or combination chemotherapy with a placebo vaccine (n=7) consisting of equivalent amounts of HA and CMC to the ACV (81). The chemotherapy protocol consisted of L-asparaginase, vincristine, cyclophosphamide, doxorubicin, lomustine, and prednisone. Chemotherapy was administered on weeks 1, 2, 3, 4, 7, 10, 13, 16, and 19, and oral prednisone was continued throughout; ACV or placebo doses were administered as intradermal injections on weeks 4, 5, 6, 7, 12, 16, 20, and 24. The median time to first progression was significantly longer in the ACV group (304 days), compared to the placebo group (41 days; p=0.0004). At clinical relapse, dogs were given a standardized rescue chemotherapy protocol, and the median second remission duration was significantly longer in the immunochemotherapy group compared to the placebo group (127 vs. 32 days; p=00167). There was no difference in the type, frequency, and severity of adverse events between treatment groups, and no difference in the quality of life based on owner survey results collected at each visit. At the conclusion of the study, dogs had DTH skin tests, and all vaccinated dogs showed a positive response, whereas no response was observed in the placebo dogs. These results further support the use of chemoimmunotherapy for treating canine lymphoma and show the ACV did not contribute to adverse events beyond those expected from chemotherapy. Because these dogs were all treated with prednisone, a potent inhibitor of lymphocytes, it raises a question about whether outcomes could be improved in dogs receiving little or no prednisone as part of their chemoimmunotherapy protocol.

The phenomenon of a prolonged second remission with rescue chemotherapy after chemoimmunotherapy has been reported in canine lymphoma patients treated with a DC ACV comprised of CD40-activated B cells loaded with tumor RNA (68). Dogs were treated with a 20-week chemotherapy protocol (cyclophosphamide, doxorubicin, vincristine, and prednisone). Those in complete remission two weeks after completing chemotherapy received the DC ACV as intradermal injections every 2 to 3 weeks for a total of 3 doses, beginning 3 to 4 weeks after completion of chemotherapy. The initial median time to disease progression was similar between the chemotherapy only (327 days; n=46) and the chemoimmunotherapy dogs (366 days, n=15). At relapse, dogs were treated with rescue chemotherapy (cyclophosphamide, vincristine, and prednisone), and only 3 out of 39 (7.7%) dogs in the chemotherapy group had a durable second remission (defined as >22 mo.), compared to 10 out of 15 (40%) dogs in the chemoimmunotherapy group. This mirrors the experience in human medicine, where patients with castration-resistant prostate cancer and advanced small cell lung cancer treated with DC ACV had unexpectedly high response rates to subsequent salvage therapy, despite a lack of improvement in initial progression-free survival from the DC ACV (82, 83). Given the positive effects of chemotherapy on the immune system relative to tumor immunology, namely depletion of T_reg that contribute to local tumor immunosuppression, chemoimmunotherapy may have broad application to other solid tumors.

Using ACV with immune checkpoint inhibitors (CPI) is another logical therapeutic strategy. ACV are used to stimulate an effector T cell response, whereas CPI are used to overcome local tumor-driven immunosuppression, allowing CD8⁺ T cells to interact effectively and kill cancer cells. The effectiveness of this approach was demonstrated in a pilot study of dogs with high-grade glioma treated with a tumor lysate ACV in combination with the CPI peptide CD200AR-L (84). In this study, 20 dogs underwent craniotomy for surgical removal of their tumor. The vaccine protocol consisted of an intradermal injection of CD200AR-L, then 24 hours later imiquimod was applied topically to the injection site; and 15 minutes later an intradermal injection of ACV mixed with CD200AR-L was given. The first dose of vaccine was administered 10 days after surgery, then repeated weekly for 3 doses, every 4 weeks for 3 doses, and every 6-8 weeks until tumor progression was noted, or death occurred. Outcomes were compared to historical controls that had similar surgeries and were treated with only the ACV (n=15). The overall median survival time of dogs treated with ACV and CD200AR-L was 12.9 months, compared to 6.8 months for dogs treated with ACV alone. The 2-year survival rate in dogs treated with ACV and CD200AR-L was 30%, and in 5 dogs with residual disease, regression was documented at the 4-month MRI imaging. In the control group, no dogs survived longer than 7 months, and regression of residual disease was not observed. Tumor progression was the cause of death in 60% of dogs treated with ACV and CD200AR-L, compared to 87% of dogs in the control group. The median survival time for high-grade gliomas in dogs treated with surgery alone is 66 days (85). These results show that ACV in the adjuvant setting increases the median survival time more than 3x from surgery alone. The addition of a canine specific CPI to the ACV protocol nearly doubled the median survival time, compared to dogs treated with the vaccine alone, with a small subset of dogs (15%) still alive more than 2 years after surgery. The USDA-CVB has recently granted a conditional license for gilvetmab (Merck Animal Health, Rahway, NJ, USA), which is a caninized monoclonal antibody against PD-1, and the first veterinary CPI (86). With the full licensure and commercialization of gilvetmab, veterinarians in the US will have the ability to treat cancer-bearing dogs with a CPI and ACV. Per the March 2024 Notice from USDA-CVB, autologous therapeutic biologics cannot be sold as experimental products after June 30, 2025 (87).

A recently described approach to canine oral malignant melanoma is to combine radiotherapy with a dendritic cell/autologous tumor cell fusion vaccine (88). Though the initial studies established safety, survival rates were not improved when compared to retrospective data for dogs receiving carboplatin without wide-margin surgery or radiotherapy.

Challenges

The studies discussed herein clearly demonstrate the potential application of ACV in veterinary cancer patients. As with all cancer therapies, not every patient benefits from ACV treatment, even within the above cited publications, and there are additional examples of veterinary ACV studies that fail to demonstrate a clear clinical benefit (57, 89-92). Predicting which patients are most likely to benefit from a given cancer therapy is a worthy goal. In the case of ACV, it is not a straightforward task given the large number of variables regarding ACV preparation (tissue handling, cell inactivation method, and adjuvant strategy), dosing (tumor antigen and adjuvant amount, route, and frequency), tumor characteristics (genetic heterogeneity, NeoAg expression, TAA expression, and extent of local immunosuppression from T_reg and tumor microenvironment) and patient characteristics (T cell fitness, and other factors that affect the ability to mount an effective immune response). This is further complicated by an incomplete knowledge of host and tumor immunology as well as a lack of validated tools to study the immune response in dogs, cats, and horses with sufficient detail. Although skin testing for DTH can provide evidence that a patient has developed cell-mediated immunity against antigens within the ACV, it is not uniformly present after ACV administration and does not always correlate with clinical response (91, 93). Although humoral antibody response to ACV correlates with treatment response (94), it is not a useful predictive test. The frequency of circulating T_reg, C reactive protein:albumin ratio, and lymphocyte:monocyte ratio may also be predictive of ACV response in dogs (92, 95). However, it is unclear if the prognostic value of these tests can be generalized to other ACV formulations, various cancer types, and different species.

ACV represent a unique cancer vaccine strategy. By virtue of using cancer tissue from the patient, ACV are likely to contain immunologically relevant antigens that can be presented to the host’s immune system at a site distant to the primary tumor with its immunosuppressive microenvironment. In addition to the therapeutic potential of ACVs they are generally well tolerated regardless of cancer type, formulation, or species (40-42, 48, 52-55, 59, 64, 96). Despite the potential therapeutic utility of ACV, they are not readily available. Commercial development of veterinary ACV faces several challenges. ACVs are created individually for each animal, rather than bulk manufacturing of a biologic, which has two major consequences. First, innovation and clinical development of ACV is being led by small animal health companies that may lack the resources to support multiple ongoing clinical trials, so they are typically focused on completing studies for regulatory approval. Second, these types of products fall outside the usual regulatory guidelines for veterinary biologics in the USA, so a special approval process has been created. This path currently includes an experimental therapeutic license and terminates in a special Autologous Prescription Product license, renewable every 2 years. Lastly, the interpretation of clinical outcome data is challenging with ACV. Even with a consistent manufacturing process, it is important to remember that each vaccine is unique, owing to the genetic and antigenic heterogeneity that exists among cancers with the same microscopic appearance. This means in a clinical study of ACVs, each patient is receiving a unique treatment, and each patient is likely to have underlying differences in their immunocompetence; therefore, caution in interpreting results is warranted. Tools that allow for stratifying subjects based on genetic characteristics of their tumor (e.g., tumor mutational burden) or immunocompetence (e.g., T cell fitness) will allow for the design of more robust clinical trials.