Cancer Immunotherapy.

The enhanced understanding of immunology experienced over the last 5 decades afforded through the tools of molecular biology has recently translated into cancer immunotherapy becoming one of the most exciting and rapidly expanding fields. Human cancer immunotherapy is now recognized as one of the pillars of treatment alongside surgery, radiation, and chemotherapy. The field of veterinary cancer immunotherapy has also rapidly advanced in the last decade with a handful of commercially available products and a plethora of investigational cancer immunotherapies, which will hopefully expand our veterinary oncology treatment toolkit over time.

Chronic adrenergic stress and generation of myeloid-derived suppressor cells: Implications for cancer immunotherapy in dogs.

Recent studies have highlighted a key role played by the sympathetic nervous system (SNS) and adrenergic stress in mediating immune suppression associated with chronic inflammation in cancer and other diseases. The connection between chronic SNS activation, adrenergic stress and immune suppression is linked in part to the ability of catecholamines to stimulate the bone marrow release and differentiation of myeloid-derived suppressor cells (MDSC). Rodent model studies have revealed an important role for ß-adrenergic receptor signalling in suppression of cancer immunity in mice subjected to chronic stresses, including thermal stress. Importantly, therapeutic blockade of beta-adrenergic responses by drugs such as propranolol can partially reverse the generation and differentiation of MDSC, and partly restore tumour immunity. Clinical trials in both humans and dogs with cancer have demonstrated that propranolol blockade can improve responses to radiation therapy, cancer vaccines and immune checkpoint inhibitors. Thus, the SNS stress response has become an important new target to relieve immune suppression in cancer and other chronic inflammatory conditions.

Outcomes from an anti-gonadotropin-releasing hormone immunotherapeutic trial in large flying foxes (Pteropus vampyrus).

OBJECTIVE: The anti-GnRH immunotherapeutic product Improvest was administered to intact male large flying foxes (Pteropus vampyrus) under managed care for androgen mitigation, leading to a decrease in agonistic behaviors, falls, and injuries from conspecific attention. ANIMALS: 12 males were included in this study. PROCEDURES: Eleven bats received subcutaneous (SC) Improvest interscapular, and 1 animal received Improvest SC in its leg. Assessments included clinical presentation, treatment, behavior, and urine and fecal glucocorticoid metabolites and testosterone (T5) concentrations. RESULTS: Eleven of the 12 bats developed reactions, which included facial edema, localized irritation, swelling of the head and neck, and pruritus with varying degrees of skin ulceration and subsequent necrosis. Three of the animals required extensive treatments, and the 1 animal who received the injection in its leg was unaffected. Posttreatment, fecal glucocorticoid metabolite and/or T5 values were at or below the nonbreeding season baseline for 3 successive breeding seasons, and there was a reduction in agonistic interactions, falls, and injuries. CLINICAL RELEVANCE: A behavioral characteristic of this species is to focus on areas of irritation that exacerbated the extent of the skin wounds. Some cases required medical, surgical, and behavioral intervention. Large flying foxes may be particularly sensitive to this immunotherapeutic when given subcutaneously in the interscapular region. Despite this reaction, the positive long-term effects on behavior and multiyear reduction of hormones suggest that the use of this immunotherapeutic warrants further investigation, although the results should be taken into consideration with other factors such as handling, treatments, chronicity of lesions.

Developing a translational murine-to-canine pathway for an IL-2/agonist anti-CD40 antibody cancer immunotherapy.

Human and canine sarcomas are difficult to treat soft tissue malignancies with an urgent need for new improved therapeutic options. Local recurrence rates for humans are between 10%-30%, and 30%-40% develop metastases. Outcomes for dogs with sarcoma vary with grade but can be similar. Pet dogs share the human environment and represent human cancer with genetic variation in hosts and tumours. We asked if our murine studies using genetically identical mice and cloned tumour cells were translatable to larger, genetically diverse domestic dogs with naturally occurring tumours, to (i) develop a canine cancer therapeutic, and (ii) to use as a translational pathway to humans. Our murine studies showed that intra-tumoral delivery of interleukin-2 (IL-2) plus an agonist anti-CD40 antibody (Ab) induces long-term curative responses ranging from 30% to 100%, depending on tumour type. We developed an agonist anti-canine-CD40 Ab and conducted a phase I dose finding/toxicology 3 + 3 clinical trial in dogs (n = 27) with soft tissue sarcomas on account of suitability for intratumoral injection and straightforward monitoring. Dogs were treated with IL-2 plus anti-CD40 antibody for 2 weeks. Three dose levels induced tumour regression with minimal side effects, measured by monitoring, haematological and biochemical assays. Importantly, our mouse and canine studies provide encouraging fundamental proof-of-concept data upon which we can develop veterinary and human immunotherapeutic strategies.

Detection of indoleamine 2,3-dioxygenase 1-expressing cells in canine normal and tumor tissues.

Cancer immunotherapy is a novel cancer treatment for canine tumors. Indoleamine 2,3-dioxygenase 1 (IDO1) is overexpressed in some human tumors and inhibits antitumor immunity. In this study, we comprehensively evaluated expression pattern of IDO1 and the nature of IDO1-expressing cells in canine normal and tumor tissues. In normal tissue samples, IDO1 expression was detected only in the lymph nodes, spleen, tonsil tissues, and colon tissues. In contrast, IDO1-positive tumor cells were observed in several tumor tissue types. This is the first study to evaluate IDO1 expression in canine normal and tumor tissues, and the results suggest that IDO1 is a promising target for novel cancer immunotherapy in dogs with tumors.

There and back again: Translating adoptive cell therapy to canine cancer and improving human treatment.

Adoptive cell transfer (ACT) is a burgeoning therapeutic modality within human immuno-oncology. Novel approaches towards ACT are being developed in the pre-clinical setting faster than they can be evaluated in human clinical trials. Many of the therapeutic approaches used in human medicine have already been evaluated to some degree in canine patients. While this form of immunotherapy in veterinary medicine is still in its infancy, as these approaches develop, canine ACT will become a tool for both the veterinary oncologist and the translational researcher. This review details canine ACT trials to date, with attention given to the precedents provided by human oncology.

Immunohistochemical Analysis of Programmed Death-Ligand 1 Expression in Equine Sarcoids.

The aim of the study was to assess the expression of the immune checkpoint inhibitor programmed death-ligand 1 (PD-L1) in equine sarcoids (ES). Programmed death-ligand 1 is expressed by various cancer cells to block T cell-mediated elimination of tumor cells. Antibodies targeting human PD-L1 were tested by immunohistochemistry for their cross-reactivity with equine PD-L1 using formalin-fixed, paraffin-embedded tissues. Our results do not support an important role of PD-L1-mediated immune evasion in ES disease and hence do not offer a rationale for anti-PD-1/PD-L1-based immunotherapy against ES.

A pilot clinical study of the therapeutic antibody against canine PD-1 for advanced spontaneous cancers in dogs.

Inhibition of programmed death 1 (PD-1), expressed on activated T cells, can break through immune resistance and elicit durable responses in human melanoma as well as other types of cancers. Canine oral malignant melanoma is one of the most aggressive tumors bearing poor prognosis due to its high metastatic potency. However, there are few effective treatments for the advanced stages of melanoma in veterinary medicine. Only one previous study indicated the potential of the immune checkpoint inhibitor, anti-canine PD-L1 therapeutic antibody in dogs, and no anti-canine PD-1 therapeutic antibodies are currently available. Here, we developed two therapeutic antibodies, rat-dog chimeric and caninized anti-canine PD-1 monoclonal antibodies and evaluated in vitro functionality for these antibodies. Moreover, we conducted a pilot study to determine their safety profiles and clinical efficacy in spontaneously occurring canine cancers. In conclusion, the anti-canine PD-1 monoclonal antibody was relatively safe and effective in dogs with advanced oral malignant melanoma and other cancers. Thus, our study suggests that PD-1 blockade may be an attractive treatment option in canine cancers.

Profile of oncological diseases in dogs and clinical effectiveness of autovaccine therapy of papillomatosis / Perfil de doenças oncológicas em caninos e eficácia clínica da terapia com autovacina em papilomatose

Papillomatosis is one of the few diseases in which autovaccine therapy is recommended, especially in the case of mass manifestation in animals. It is noted that papillomas are diagnosed in 15.7% of cases in histological diagnosis of oncological diseases. Therefore, this work studied the profile of oncological diseases in dogs and the clinical effectiveness of autovaccine therapy of papillomatosis. The article gives a morphological description of papillomas used as a biomaterial, and also describes the technological methods of manufacturing an autovaccine used to treat animals against papillomatosis. A therapeutic scheme for administering the vaccine was proposed and tested, and the results of the clinical trial are presented, as well as a reflection on some moments of the mechanism of vaccine therapy for papillomatosis.(AU)

A papilomatose é uma das poucas doenças onde se recomenda a terapia com autovacina, principalmente no caso de manifestação em massa em animais. Nota-se que papilomas são diagnosticados em 15,7% dos casos em diagnóstico histológico de doenças oncológicas. Portanto, este trabalho estudou o perfil das doenças oncológicas em cães e a eficácia clínica da terapia com autovacina em papilomatose. O presente artigo fornece descrição morfológica dos papilomas utilizados como biomaterial, além da descrição dos métodos tecnológicos de fabricação de uma autovacina usada para tratar animais contra a papilomatose. Um esquema terapêutico para a administração da vacina foi proposto e testado, e os resultados do ensaio clínico são apresentados, e é feita uma reflexão sobre alguns momentos do mecanismo da terapia de vacina para papilomatose.(AU)

Sarcomas-A barren immunological wasteland or field of opportunity for immunotherapy?

Key advances in our understanding of immunobiology and the immunosuppressive mechanisms of the tumour microenvironment have led to significant breakthroughs in manipulating the immune system to successfully treat cancer. Remarkable therapeutic responses have occurred with tumours that carry a high mutational burden. In these cases, pre-existing tumour-specific T cells can be rejuvenated via checkpoint inhibition to eliminate tumours. Furthermore, durable remissions have been achieved in haematological malignancies following adoptive transfer of T cells that specifically target cell surface proteins where expression is restricted to the malignancy's cell of origin. Soft tissue sarcomas and bone sarcomas have a paucity of non-synonymous somatic mutations and do not commonly express known, targetable, tumour-specific antigens. Historically, soft tissue sarcomas have been considered immunologically 'cold' and as such, unlikely candidates for immune therapy. Here, we review the immune landscape of canine and feline sarcomas and the immunotherapeutic strategies that have been employed in veterinary clinical trials to improve patient outcome. We also provide insight into immunotherapeutic approaches being used to treat human sarcomas. Together, current data indicates that, rather than a barren immunological wasteland, sarcomas represent a field of opportunities for immunotherapies. Furthermore, we and others would suggest that strategic combinations of immunotherapeutic approaches may hold promise for more effective treatments for high grade soft tissue sarcomas and bone sarcomas.

Emerging therapeutic approaches for canine sarcomas: Pushing the boundaries beyond the conventional.

Sarcomas represent a group of genomically chaotic, highly heterogenous tumours of mesenchymal origin with variable mutational load. Conventional therapy with surgery and radiation therapy is effective for managing small, low-grade sarcomas and remains the standard therapeutic approach. For advanced, high-grade, recurrent, or metastatic sarcomas, systemic chemotherapy provides minimal benefit, therefore, there is a drive to develop novel approaches. The discovery of "Coley's toxins" in the 19th century, and their use to stimulate the immune system supported the application of unconventional therapies for the treatment of sarcomas. While promising, this initial work was abandoned and treatment paradigm and disease course of sarcomas was largely unchanged for several decades. Exciting new therapies are currently changing treatment algorithms for advanced carcinomas and melanomas, and similar approaches are being applied to advance the field of sarcoma research. Recent discoveries in subtype-specific cancer biology and the identification of distinct molecular targets have led to the development of promising targeted strategies with remarkable potential to change the landscape of sarcoma therapy in dogs. The purpose of this review article is to describe the current standard of care and limitations as well as emerging approaches for sarcoma therapy that span many of the most active paradigms in oncologic research, including immunotherapies, checkpoint inhibitors, and drugs capable of cellular metabolic reprogramming.

In silico identification of immunotherapeutic and diagnostic targets in the glycosylphosphatidylinositol metabolism of the coccidian Sarcocystis aucheniae.

Meat of the South American camelids (SACs) llama and alpaca is an important source of animal protein and income for rural families in the Andes, and a product with significant growth potential for local and international markets. However, infestation with macroscopic cysts of the coccidian protozoon Sarcocystis aucheniae, a parasitosis known as SAC sarcocystosis, significantly hampers its commercialization. There are no validated methods to diagnose the presence of S. aucheniae cysts other than carcass examination. Moreover, there are no available drugs or vaccines to cure or prevent SAC sarcocystosis. Identification of relevant molecules that act at the host-pathogen interface can significantly contribute to the control of this disease. It has been shown for other pathogenic protozoa that glycosylphosphatidylinositol (GPI) is a critical molecule implicated in parasite survival and pathogenicity. This study focused on the identification of the enzymes that participate in the S. aucheniae GPI biosynthetic pathway and the repertoire of the parasite GPI-anchored proteins (GPI-APs). To this aim, RNA was extracted from parasite cysts and the transcriptome was sequenced and translated into amino acid sequences. The generated database was mined using sequences of well-characterized GPI biosynthetic enzymes of Saccharomyces cerevisiae and Toxoplasma gondii. Eleven enzymes predicted to participate in the S. aucheniae GPI biosynthetic pathway were identified. On the other hand, the database was searched for proteins carrying an N-terminal signal peptide and a single C-terminal transmembrane region containing a GPI anchor signal. Twenty-four GPI-anchored peptides were identified, of which nine are likely S. aucheniae-specific, and 15 are homologous to membrane proteins of other coccidians. Among the latter, 13 belong to the SRS domain superfamily, an extensive group of coccidian GPI-anchored proteins that mediate parasite interaction with their host. Phylogenetic analysis showed a great degree of intra- and inter-specific divergence among SRS family proteins. In vitro and in vivo experiments are needed to validate S. aucheniae GPI biosynthetic enzymes and GPI-APs as drug targets and/or as vaccine or diagnostic antigens.

Vaccine strain Listeria monocytogenes abscess in a dog: a case report.

BACKGROUND: Listeria monocytogenes is a promising therapeutic vaccine vector for cancer immunotherapy. Although highly attenuated, three cases of systemic listeriosis have been reported in people following treatment with Listeria-based therapeutic vaccines. This complication has thus far not been reported in canine patients. CASE PRESENTATION: A dog previously diagnosed with osteoblastic osteosarcoma was presented for care following administration of three doses of the Canine Osteosarcoma Vaccine-Live Listeria Vector. On routine staging chest radiographs, mild sternal lymphadenopathy and a right caudoventral thoracic mass effect were noted. Further evaluation of the mass effect with computed tomography and ultrasound revealed a cavitated mass associated with the 7th right rib. Aspirates of the mass cultured positive for Listeria monocytogenes. The mass and associated ribs were surgically removed. Histopathology was consistent with metastatic osteoblastic osteosarcoma. Treatment was continued with doxorubicin chemotherapy and at the time of publication, the dog was alive over 1 year following diagnosis with no evidence of further disease progression. Genotyping of the abscess-derived L. monocytogenes was consistent with the vaccine strain. CONCLUSIONS: This case represents the first veterinary case to describe development of a Listeria abscess following administration of a Listeria-based therapeutic vaccine.

Lymphokine-activated killer cell transplantation after anti-cancer treatment in two aged cats.

Immunotherapy improves both survival and remission rates after cancer surgery in humans, but its veterinary use has been limited. We determined the safety and feasibility of lymphokine-activated killer (LAK) cell transplantation in two aged cats that had undergone surgery for malignancy. Case 1 involved an 18-year-old male Japanese domestic cat. The cat exhibited appetite loss and poor physical activity after the surgical excision of oral squamous cell carcinoma followed by four sessions of radiotherapy, and the owner strongly requested immunotherapy for preventing further deterioration in the animal's quality of life (QOL). We subsequently administered LAK cells three times during a 2-month period. Case 2 involved a 20-year-old female Japanese domestic cat who had undergone mammectomy after a diagnosis of breast adenocarcinoma. The owner strongly requested immunotherapy for QOL maintenance. We administered LAK cells four times over a period of 5 months. Autologous peripheral blood mononuclear cells (PBMCs) fractionated using density gradient centrifugation were cultured in the media containing a high concentration of interleukin-2 and supplemented with 2.5% fetal calf serum. The derived LAK cells were centrifuged, suspended in 10 ml of saline containing 1% of the subject's own blood, and infused into the cephalic vein of the cats over 30 min. The composition ratios of CD3, CD4, CD8, and CD21 were evaluated by flow cytometry. Bacterial culture and endotoxin testing for a sample of LAK cells showed negative results in both the cases. The leukocyte and erythrocyte counts and the body temperature were assessed on days 7, 14, and 21 after the transfusion. No abnormal signs were observed in either case, which confirmed the safety of the procedure. QOL scores showed no significant changes after the treatment, and the body temperature remained steady throughout the treatment. The findings from these cases suggest that the transplantation of LAK cells derived from PBMCs may be safe and feasible for use in cats, regardless of their age.

Cancer Immunotherapies.

The enhanced understanding of immunology experienced over the last 4 decades afforded through the tools of molecular biology has recently translated into cancer immunotherapy becoming one of the most exciting and rapidly expanding fields. Human cancer immunotherapy is now recognized as one of the pillars of treatment alongside surgery, radiation, and chemotherapy. The field of veterinary cancer immunotherapy has also rapidly advanced in the last decade with a handful of commercially available products and a plethora of investigational cancer immunotherapies that will hopefully expand the veterinary oncology treatment toolkit over time.

Novel Treatments for Lymphoma.

Lymphoma is a common disease in companion animals. Although conventional chemotherapy has the potential to induce remission and prolong life, relapse is common, and novel treatments are needed to improve outcome. This review discusses recent modifications/adjustments to conventional standard of care therapy for canine and feline lymphoma, as well as cutting-edge immunotherapy and small-molecule-based approaches that are in varying stages of regulatory approval.

Safety Evaluation of Autologous Tissue Vaccine Cancer Immunotherapy in a Canine Model.

BACKGROUND/AIM: Previous work in rodent models showed that an autologous tissue vaccine is both a safe and effective approach for treating cancer; however, as a translational step, safety must first be evaluated in a more clinically-relevant model. MATERIALS AND METHODS: An autologous immunotherapy produced from resected tumors, was evaluated in a clinically-relevant canine model to assess safety. Ninety-three dogs with spontaneously occurring tumors received vaccination with inactivated autologous tumor tissue combined with an adjuvant of particulate porcine small intestinal submucosa extracellular matrix (SIS-ECM). Patients were followed to assess the occurrence of adverse events, overall survival, and tumor recurrence and/or metastasis. RESULTS: A small number (12%) of patients experienced limited, mild pyrexia, injection site swelling, or lethargy, all resolving without clinical intervention. CONCLUSION: Autologous whole cell cancer immunotherapy can be used safely in the canine model of cancer and represents a safe approach for the treatment for cancer.

Immunotherapy for the treatment of canine transmissible venereal tumor based in dendritic cells pulsed with tumoral exosomes.

Context: Exosomes secreted by tumor cells are a good source of cellular components that stimulate the immune response, such as alarmins (mRNA, tetraspanins (CD9, CD63, CD81), heat-shock proteins, major histocompatibility complex class I molecules) and tumor-associated antigens. These properties permit to pulsed dendritic cells in the immunotherapy for many cancers types. The aim of this study was to demonstrate the use of exosomes derived from canine transmissible venereal tumor (CTVT) as an antigen to pulsed dendritic cells and its administration in dogs with CTVT as treatment against this disease. Material and methods: From primary culture of CTVT cells the exosomes were isolated and characterized by scanning electron microscopy assay, dot blot and protein quantification. The monocytes of each patient were differentiated to dendritic cells (DC) and pulsed with CTVT exosomes (CTVTE). Phagocytosis, tumor size, populations of lymphocytes and IFN-c levels were evaluated. Results: The CTVTE showed a size around 90 nm. CD81, CD63, CD9 and Hsp70 were expressed. Monocytes showed an expression of 85.71% for CD14+, 12.3% for CD80+, 0.1% for CD83+ and 0.8% for DLA-II. In DC 5.1% for CD14+, 86.7% for CD80+, 90.1% for CD83+ and 92.6% for DLA-II and a phagocytosis of 63% was obtained by FITC Dextran test. No side effects were observed in the experimental groups with our therapy. Tumor regression was of 100% at the seventh week, as well as an increase in the level of IFN-γ (142 pg/ml), and CD4+ (28%) and CD8+ (34%) cell percentage. Discusion and conclusion: These results have shown that DC pulsed with tumor exosomes induce regression of the TVT in dogs.

Immunogenic potential of three transmissible venereal tumor cell lysates to prime canine-dendritic cells for cancer immunotherapy.

Whole tumor cell lysates consist of a mixture of tumor antigens and danger associated molecular patterns (DAMPs) that can be used for dendritic cell maturation and consequently for the activation of a polyclonal T cell-specific tumor response. We evaluated the in vitro efficacy of three different preparations of canine transmissible venereal tumor (TVT) cell lysates: hypochlorous acid-whole tumor cell lysates (HOCl-L), heat shock-whole tumor cell lysates (HS-L), and freeze-thaw cycles-whole tumor cell lysates (FT-L) for the maturation of canine-derived dendritic cells. Our results showed calreticulin, HSP70, and HSP90 release in the three tumor lysates preparations (HOCl-L, HS-L, and FT-L); however, HMGB1 was detected only in HOCl-L and FT-L. Additionally, the uptake by HOCl-L pulsed dendritic cell (DC) increased compared to HS-L and FT-L pulsed DC; and dendritic cell maturation was confirmed by the appropriate cell surface markers (CD11c, CD80, CD83, and MHCII). Furthermore, dendritic cells pulsed with HOCl-L, HS-L or FT-L were cultured with canine lymphocytes. There was an increase of Th1-type cytokines (IL-12, TNF-α, and IFN-γ), in all the tumor cell lysates co-cultures, this correlates with T lymphocyte activation and cytotoxic response. Our data confirm that TVT cell lysates can induce functional canine-DC and that HOCl-L is the most effective one. This preparation of TVT cell lysates with HOCl is an attractive approach that allows the recognition of neoantigens as potential tumor targets and DC priming and therefore could be used for cancer immunotherapy against TVT.