IntroductionNatural killer (NK) cells in mice and humans are key effectors of the innate immune system with complex immunoregulatory functions, and diverse subsets have been identified with distinct characteristics and roles. Companion dogs with spontaneous cancer have been validated as models of human disease, including cancer immunology and immunotherapy, and greater understanding of NK cell heterogeneity in dogs can inform NK biology across species and optimize NK immunotherapy for both dogs and people.MethodsHere, we assessed canine NK cell populations by single-cell RNA sequencing (scRNAseq) across blood, lung, liver, spleen, and placenta with comparison to human NK cells from blood and the same tissues to better characterize the differential gene expression of canine and human NK cells regarding ontogeny, heterogeneity, patterns of activation, inhibition, and tissue residence.ResultsOverall, we observed tissue-specific NK cell signatures consistent with immature NK cells in the placenta, mature and activated NK cells in the lung, and NK cells with a mixed activated and inhibited signature in the liver with significant cross-species homology.DiscussionTogether, our results point to heterogeneous canine NK populations highly comparable to human NK cells, and we provide a comprehensive atlas of canine NK cells across organs which will inform future cross-species NK studies and further substantiate the spontaneous canine model to optimize NK immunotherapy across species.

A clinical trial in dogs with spontaneous osteosarcoma was performed to assess a recombinant Listeria expressing a chimeric human HER2 (ADXS31-164c) as an adjunctive vaccine strategy to prevent metastatic disease and determine immunological correlates of clinical outcome. A total of 118 dogs with appendicular osteosarcoma were recruited into a 1-arm, multicenter, prospective trial of standard of care (SOC) therapy followed by ADXS31-164c. ADXS31-164c was well tolerated, with mostly transient, low-grade side effects. Significant differences in median disease-free interval (DFI) or median overall survival (OS) of immunized dogs compared to a historical cohort of dogs receiving SOC only were not observed. Elite survivors (DFI >490 days) showed transient increases in temperature and serum cytokines, including IL-6 and TNF-α, after the first immunization compared to short-term survivors (DFI 150-235 days). However, repeat immunizations in short-term survivors led to improved and comparable pyrexic and cytokine responses to elite survivors. PBMC transcriptomic analysis following vaccinations revealed robust cytotoxic activity in elite but not short-term survivors. Although ADXS31-164c did not significantly extend DFI or OS, immune responses to ADXS31-164c distinguished elite from short-term survivors. Improvement of immune responses over sequential ADXS31-164c administrations supports a future trial design of recurrent immunizations to improve outcomes of otherwise short-term survivors.

Background:Canine and human malignant melanoma are naturally occurring cancers with many similarities, making the dog an important parallel patient population to study both diseases. However, development of canine anti-human antibodies (CAHA) needs to be considered when evaluating humanized biotherapeutics in dogs. Objectives:Characterize CAHA in sera from dogs with spontaneous melanoma receiving radiotherapy and intratumoral immunocytokine (IT-IC) with humanized 14.18-IL2. Methods:Serum samples were obtained pre-treatment and at several post-treatment times from 12 dogs with locally advanced or metastatic melanoma treated with radiotherapy to the primary site and regional lymph nodes (when clinically involved) followed by IT-IC of humanized 14.18-IL2. Two CAHA assays were developed. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies against the humanized IgG component of hu14.18-IL2. A flow cytometry assay was developed to determine the ability of CAHA to inhibit binding of a mouse anti-GD2 monoclonal antibody to its target. Results:Post-treatment sera from 7 of 12 dogs developed CAHA levels over pre-treatment that were identified by ELISA as significant increases at Day 30 and/or Day 60. Day 10, Day 30, and Day 60 post-treatment sera from 10 of 12 dogs significantly inhibited the binding of anti-GD2 monoclonal antibody to its target compared to pre-treatment. Significant binding inhibition was also detected in 2 of 12 dogs after local RT but before IT-IC (Day 1). Normal canine sera did not mediate binding inhibition. Conclusions:This study advances CAHA detection strategies and reports the kinetics of CAHA following IT-IC in dogs with spontaneous melanoma.

IntroductionNatural killer (NK) cells have great potential to extend the promise of cancer immunotherapy, but additional research is needed to improve their efficacy in solid cancers. Dogs develop spontaneous cancers with striking similarities to humans and can serve as a crucial link to bridge murine studies and human clinical trials to improve treatment outcomes across species and identify potential biomarkers of response.MethodsUsing single-cell RNA sequencing (scRNAseq), we integrated blood, tissue, and tumor samples from dog and human donors to compare NK cell gene expression and develop a canine sarcoma infiltrating NK signature. Canine tissue and tumor NK cell signatures were then used to contextualize NK cell changes in first-in-dog immunotherapy clinical trials.ResultsTumor infiltrating NK cells from both canine and human sarcomas exhibited enhanced migration with a simultaneously exhausted signature that most closely correlated transcriptionally with NK cells isolated from the liver. We also analyzed peripheral blood NK cells from dogs on first-in-dog clinical trials undergoing three distinct NK-targeting immunotherapy regimens, observing that dogs with favorable responses demonstrated increased NK proportions posttreatment. Genes upregulated in NK cells in the peripheral blood of good responders included genes associated with activated NK cells and revealed post-treatment gene expression changes in the blood as a predictor of response.DiscussionOverall, NK effector functions are well adapted to their tissue of residence but dysregulated in sarcoma infiltrating NK cells despite enhanced migration. We describe NK cell trends across canine clinical trials as a platform through which we can elucidate mechanisms of response and determine novel immunotherapy strategies to improve cancer outcomes in both humans and dogs.

The absence of tumor-infiltrating lymphocytes negatively impacts the response to chemotherapy and prognosis in all subtypes of breast cancer. Therapies that stimulate a proinflammatory environment may help improve the response to standard treatments and also to immunotherapies such as checkpoint inhibitors. Newcastle disease virus (NDV) shows oncolytic activity, as well as immune modulating potential, in the treatment of breast cancer in vitro and in vivo; however, its potential to enhance tumor-infiltrating immune cells in breast cancer has yet to be evaluated. Since spontaneous canine mammary tumors represent a translational model of human breast cancer, we conducted this proof-of-concept study, which could provide a rationale for further investigating NDV-MLS as immunotherapy for mammary cancer. Six female companion dogs with spontaneous mammary cancer received a single intravenous and intratumoral injection of oncolytic NDV-MLS. Immune cell infiltrates were evaluated by histology and immunohistochemistry in the stromal, intratumoral, and peritumoral compartments on day 6 after viral administration. Increasing numbers of immune cells were documented post-viral treatment, mainly in the peritumoral compartment, where plasma cells and CD3+ and CD3-/CD79- lymphocytes predominated. Viral administration was well tolerated, with no significant adverse events. These findings support additional research on the use of NDV-MLS immunotherapy for mammary cancer.

ABSTRACT MAPK has been reported as a key oncogenic pathway for canine histiocytic sarcoma, which can be pharmacologically targeted with trametinib, a small inhibitor of MEK1/2. Preliminary data showed promising antitumor activity in in vitro and in vivo models and represented a proof of concept to translate the findings from bench to bedside. In this phase I, dose escalating study using a 3 + 3 cohort design, trametinib was evaluated in 18 dogs with cancer. Adverse events were graded according to VCOG‐CTCAE v2. Blood samples and tumour biopsies were collected for pharmacokinetic and pharmacodynamic assessment. Trametinib was well tolerated with a maximum tolerated dose of 0.5 mg/m 2 /day, PO. Dose‐limiting toxicities included systemic hypertension, proteinuria, lethargy and elevated ALP, and were all Grade 3. The drug exposures increased more than linearly with dose since the elimination of trametinib was saturable. At a dose of 500 μg Q24h (0.5 mg/m 2 /day in a 30 kg dog), approximately 70% of dogs had an average steady‐state concentration of 10 ng/mL, achieved after approximately 2 weeks. This threshold was associated with clinical efficacy in humans. Target engagement was not observed in biospecimens collected on Days 0 and 7. In conclusion, trametinib was considered safe in dogs with cancer, and the dose of 0.5 mg/m 2 /day was the recommended dose for phase II studies.

Canine malignant melanoma provides a clinically relevant, large animal parallel patient population to study the GD2-reactive hu14.18-IL-2 immunocytokine as it is similar to human melanoma and expresses GD2. The objectives of this study were to evaluate safety, radiation fractionation, and identify informative biomarkers of an in-situ tumor vaccine involving local radiation therapy plus intratumoral–immunocytokine in melanoma tumor-bearing dogs. Twelve dogs (six dogs/arm) with locally advanced or metastatic melanoma were randomized to receive a single 8 Gy fraction (arm A) or three 8 Gy fractions over 1 week (arm B) to the primary site and regional lymph nodes (when clinically involved) with the single or last fraction 5 days before intratumoral–immunocytokine at 12 mg/m 2 on 3 consecutive days. Serial tumor biopsies were obtained. All 12 dogs completed protocol treatment, and none experienced significant or unexpected adverse events. Evidence of antitumor activity includes one dog with a complete response at day 60, one dog with a partial response at day 60, and four dogs with mixed responses. Histology of serial biopsies shows a variably timed increase in intratumoral lymphocytic inflammation in some dogs. Canine NanoString analyses of serial biopsies identified changes in gene signatures of innate and adaptive cell types versus baseline. There were no significant differences in NanoString results between arm A and arm B. We conclude that intratumoral–immunocytokine in combination with local radiation therapy in canine melanoma is well tolerated and has antitumor activity with the potential to inform clinical development in melanoma patients.

Supplementary Table 1 contains the inclusion and exclusion criteria for pet dogs considered for enrollment into the SOC and SOC + S clinical trial arms.

Preventative anti-cancer vaccination strategies have long been hampered by the challenge of targeting the diverse array of potential tumor antigens, with successes to date limited to cancers with viral etiologies. Identification and vaccination against frameshift neoantigens conserved across multiple species and tumor histologies is a potential cancer preventative strategy currently being investigated. Companion dogs spontaneously develop cancers at a similar incidence to those in people and are a complementary comparative patient population for the development of novel anti-cancer therapeutics. In addition to an intact immune system with tumors that arise in an autochthonous tumor microenvironment, dogs also have a shorter lifespan and temporally compressed tumor natural history as compared to humans, which allows for more rapid evaluation of safety, immunogenicity, and efficacy of cancer vaccination strategies. Here we describe the study protocol for the Vaccination Against Canine Cancer Study (VACCS), the largest interventional cancer clinical trial conducted in companion dogs to date. In addition to safety and immunogenicity, the primary endpoint of VACCS is the cumulative incidence (CI) of dogs developing malignant neoplasia of any type at the end of the study period. Secondary endpoints include changes in incidence of specific tumor types, survival times following neoplasia diagnosis, and all-cause mortality.

Most primary cardiac tumors in dogs are located in the right atrium/atrial appendage, with hemangiosarcoma being the most common. The aims of this retrospective, case series were to describe outcomes for seven dogs with right atrial tumors treated with hypofractionated intensity-modulated radiotherapy and concurrent vinblastine and propranolol. One dog had a complete response, four dogs had partial responses and two dogs had stable disease after treatment. Effusions resolved in all dogs. Median progression-free survival was 290 days. Five dogs died from metastatic disease, one dog from unrelated neoplasia, and one dog is alive. Median overall survival was 326 days. Three dogs with confirmed hemangiosarcoma survived 244, 326, and 445 days. Two dogs developed clinically significant, but nonfatal, cardiac arrhythmias. One dog that received three courses of radiation had subclinical myocardial and arterial fibrosis at necropsy. Hypofractionated chemoradiotherapy was well tolerated and may provide clinical benefit in dogs with right atrial tumors.

Background Dogs are an outbred species that develop spontaneous cancers with striking similarities to humans and can serve as a crucial link to optimize immunotherapy treatments. We have completed canine immunotherapy trials using overlapping but distinct methods to stimulate natural killer (NK) cells in dogs with solid tumors. Our objective was to investigate changes in NK cell populations in response to immunotherapy and compare gene expression profiles between dogs receiving three separate NK-targeting treatments. Methods Samples were available for a total of seven dogs treated for cancer at UC Davis (UCD Trial #1, n=2, UCD Trial #2 n=1) and University of Wisconsin (UW, n=4) in IACUC-approved trials. Dogs in UCD Trial #1 received two injections of autologous NK cells in combination with inhaled rhIL-15. Dogs in UCD Trial #2 underwent palliative radiotherapy (RT) in addition to infusion of PBMC-derived allogeneic NK cells. Dogs in the UW cohort received low-dose molecular targeted radionuclide therapy (MTRT) with external beam RT and intratumoral injection of IL-2 fusion cytokine. Matched pre- and post-treatment samples were submitted for single-cell RNA sequencing and integrated, data visualization was performed using R packages, Seurat and ggplot2. We hypothesized that trials would have distinct NK-activation signatures in response to unique treatment. Results NK cells were able to be identified in each dataset by expression of NK-related genes, including NCR3, and lack of CD3 expression. Cells within the NK cluster following treatment with RT and allogeneic transfer had significantly increased expression of GZMA with an over two-fold increase in the percentage of NK cells post-treatment, reaching 14% of PBMCs. During treatment with RT and allogeneic transfer, NK cells retained steady expression of NK activating and functional genes although we also observed a distinct peak in KLRA1 expression 7 days after NK transfer. Dogs receiving MTRT therapy had increased expression of several activation markers, including CD16, NCR1, KLRA1 and CD69, in post-treatment NK cells compared to pretreatment. Notably, the MTRT therapy-specific NK gene activation signature in response to treatment was similar across patients in the cohort and distinct from dogs receiving adoptive NK transfer. Conclusions This scRNASeq analysis provides insight into the diverse mechanisms of NK activation during unique NK-targeting immunotherapy regimens in dogs with cancer. Our transcriptomic analysis of first-in-dog clinical trials serves as a basis for investigating biomarkers of NK responses in innovative canine immunotherapy combinations. Ethics Approval The animal studies were reviewed and approved by IACUC and UC Davis Clinical Trials Review Board. Written informed consent was obtained from the owners for the participation of their animals in this study.

Profiling the T cell receptor (TCR) repertoire using next-generation sequencing has become common in both human and translational research. Companion dogs with spontaneous tumors, including canine melanoma, share several features, e.g., natural occurrence, shared environmental exposures, natural outbred population, and immunocompetence. T cells play an important role in the adaptive immune system by recognizing specific antigens via a surface TCR. As such, understanding the canine T cell response to vaccines, cancer, immunotherapies, and infectious diseases is critically important for both dog and human health. Off-the-shelf commercial reagents, kits and services are readily available for human, non-human primate, and mouse in this context. However, these resources are limited for the canine. In this study, we present a cost-effective protocol for analysis of canine TCR beta chain genes. Workflow can be accomplished in 1-2 days starting with total RNA and resulting in libraries ready for sequencing on Illumina platforms.

Supplementary Table 4 contains a summary of the outcomes, with statistical comparisons, of the dogs enrolled in the SOC + sirolimus clinical trial arm. This includes the DFI and outcomes of the groups of dogs stratified by tumor location and ALP status.

Supplementary Figure Legend from Cooperative Activity of Cytotoxic Chemotherapy with Antiangiogenic Thrombospondin-I Peptides, ABT-526 in Pet Dogs with Relapsed Lymphoma

Supplementary Table 2 contains the summary comparison of both the Intent-to-treat and Per-protocol analyses of clinical outcomes for dogs enrolled in Standard of Care (SOC) and Standard of Care + sirolimus

Supplementary Figure 1 from Cooperative Activity of Cytotoxic Chemotherapy with Antiangiogenic Thrombospondin-I Peptides, ABT-526 in Pet Dogs with Relapsed Lymphoma