S6K-STING interaction regulates cytosolic DNA-mediated activation of the transcription factor IRF3.

Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.

Increased tumor-infiltrating lymphocyte density is associated with favorable outcomes in a comparative study of canine histiocytic sarcoma.

Histiocytic sarcoma (HS) is a rare and aggressive tumor in humans with no universally agreed standard of care therapy. Spontaneous canine HS exhibits increased prevalence in specific breeds, shares key genetic and biologic similarities with the human disease, and occurs in an immunocompetent setting. Previous data allude to the immunogenicity of this disease in both species, highlighting the potential for their successful treatment with immunotherapy. Quantification of CD3 tumor-infiltrating lymphocytes (TIL) in five cases of human HS revealed variable intra-tumoral T cell infiltration. Due to the paucity of human cases and lack of current model systems in which to appraise associations between anti-tumor immunity and treatment-outcome in HS, we analyzed clinical data and quantified TIL in 18 dogs that were previously diagnosed with localized HS and treated with curative-intent tumor resection with or without adjuvant chemotherapy. As in humans, assessment of TIL in biopsy tissues taken at diagnosis reveal a spectrum of immunologically "cold" to "hot" tumors. Importantly, we show that increased CD3 and granzyme B TIL are positively associated with favorable outcomes in dogs following surgical resection. NanoString transcriptional analyses revealed increased T cell and antigen presentation transcripts associated with prolonged survival in canine pulmonary HS and a decreased tumor immunogenicity profile associated with shorter survivals in splenic HS. Based on these findings, we propose that spontaneous canine HS is an accessible and powerful novel model to study tumor immunology and will provide a unique platform to preclinically appraise the efficacy and tolerability of anti-cancer immunotherapies for HS.

Applications and Opportunities for Immune Cell CAR Engineering in Comparative Oncology.

Chimeric antigen receptor (CAR) T-adoptive cell therapy has transformed the treatment of human hematologic malignancies. However, its application for the treatment of solid tumors remains challenging. An exciting avenue for advancing this field lies in the use of pet dogs, in which cancers that recapitulate the biology, immunological features, and clinical course of human malignancies arise spontaneously. Moreover, their large size, outbred genetic background, shared environment with humans, and immunocompetency make dogs ideal for investigating and optimizing CAR therapies before human trials. Here, we will outline how challenges in early clinical trials in patients with canine lymphoma, including issues related to autologous CAR T-cell manufacturing, limited CAR T-cell persistence, and tumor antigen escape, mirrored challenges observed in human CAR T trials. We will then highlight emerging adoptive cell therapy strategies currently under investigation in dogs with hematological and solid cancers, which will provide crucial safety and efficacy data on novel CAR T regimens that can be used to support clinical trials. By drawing from ongoing studies, we will illustrate how canine patients with spontaneous cancer may serve as compelling screening platforms to establish innovative CAR therapy approaches and identify predictive biomarkers of response, with a specific emphasis on solid tumors. With increased funding for canine immunotherapy studies, multi-institutional investigations are poised to generate highly impactful clinical data that should translate into more effective human trials, ultimately benefiting both human and canine cancer patients.

Malignant transformation and subsequent leptomeningeal carcinomatosis of a gastric polyp in a dog.

Progressive carcinogenesis of a gastric polyp with transformation to gastric adenocarcinoma and subsequent development of leptomeningeal carcinomatosis is described in an adult male Scottish terrier. Presenting clinical signs consisted of vomiting with intermittent hematemesis. Surgical biopsies over the course of 14 months documented the progression from gastric polyp to minimally invasive gastric carcinoma to invasive gastric adenocarcinoma, a pathogenesis not previously documented in veterinary oncology. The patient ultimately developed neurologic pathology and was euthanized, and necropsy evaluation identified widespread carcinomatosis with accompanying leptomeningeal metastasis. As in humans, gastric polyps in dogs rarely have malignant potential.

Quantification of CD3, FoxP3, and granzyme B immunostaining in canine renal cell carcinoma.

Tumor-infiltrating lymphocyte (TIL) density plays an important role in anti-tumor immunity and is associated with patient outcome in various human and canine malignancies. As a first assessment of the immune landscape of the tumor microenvironment in canine renal cell carcinoma (RCC), we retrospectively analyzed clinical data and quantified CD3, FoxP3, and granzyme B immunostaining in formalin-fixed paraffin-embedded tumor samples from 16 dogs diagnosed with renal cell carcinoma treated with ureteronephrectomy. Cell density was low for all markers evaluated. Increased numbers of intratumoral FoxP3 labelled (+) cells, as well as decreased granzyme B+: FoxP3+ TIL ratio, were associated with poor patient outcomes. Our initial study of canine RCC reveals that these tumors are immunologically cold and Tregs may play an important role in immune evasion.

Calculation of dose intensity and comparison of published methods using a cohort of canine T-cell lymphoma patients undergoing CHOP-based chemotherapy.

Methods of calculating and reporting dose intensity (DI) of CHOP-based protocols in the veterinary literature vary. The goal of this retrospective study is to examine the prognostic significance of the average percentage of planned DI received in a cohort of canine T-cell lymphoma patients treated with a modified CHOP protocol with corresponding toxicity and efficacy data. Our data set of 40 dogs was analysed using various previously published methods for calculating DI. Median progression-free survival and overall survival were 91 and 196 days, respectively. Receiving a higher percentage of planned DI was not found to be associated with patient outcome. Outcomes remain poor for dogs with T-cell lymphoma treated with CHOP-based chemotherapy irrespective of received DI. Standard methods of DI calculation and reporting should be adopted in veterinary oncology to enable repeatable and rigorous comparisons of published chemotherapy protocols and to ascertain the potential prognostic relevance of DI in canine lymphoma patients.

Evaluation of mechlorethamine, vinblastine, procarbazine, and prednisone for the treatment of resistant multicentric canine lymphoma.

Multi-agent chemotherapy successfully induces remission in most naïve, high-grade canine lymphoma patients; however, disease recurrence is common. MOPP (mechlorethamine, vincristine, procarbazine, and prednisone) is an effective rescue protocol used to re-induce remission, but is associated with gastrointestinal toxicity and can be a less desirable option for patients that previously failed vincristine-containing protocols. Therefore, alternative members of the vinca alkaloid family, such as vinblastine, could be potentially advantageous as substitutes for vincristine to reduce gastrointestinal toxicity and chemoresistance. The objective of this study was to report the clinical outcomes and toxicity of 36 dogs with relapsed or refractory multicentric lymphoma treated with a modified MOPP protocol whereby vincristine was replaced with vinblastine (MVPP). The overall response rate to MVPP was 25% with a median progression free survival of 15 days and a median overall survival of 45 days. MVPP at the prescribed doses resulted in modest and transient clinical benefit, but was well tolerated with no treatment delays or hospitalizations secondary to side effects. Given the minimal toxicity, dose intensification could be considered to improve clinical responses.

Unedited allogeneic iNKT cells show extended persistence in MHC-mismatched canine recipients.

Allogeneic invariant natural killer T cells (allo-iNKTs) induce clinical remission in patients with otherwise incurable cancers and COVID-19-related acute respiratory failure. However, their functionality is inconsistent among individuals, and they become rapidly undetectable after infusion, raising concerns over rejection and limited therapeutic potential. We validate a strategy to promote allo-iNKT persistence in dogs, an established large-animal model for novel cellular therapies. We identify donor-specific iNKT biomarkers of survival and sustained functionality, conserved in dogs and humans and retained upon chimeric antigen receptor engineering. We reason that infusing optimal allo-iNKTs enriched in these biomarkers will prolong their persistence without requiring MHC ablation, high-intensity chemotherapy, or cytokine supplementation. Optimal allo-iNKTs transferred into MHC-mismatched dogs remain detectable for at least 78 days, exhibiting sustained immunomodulatory effects. Our canine model will accelerate biomarker discovery of optimal allo-iNKT products, furthering application of MHC-unedited allo-iNKTs as a readily accessible universal platform to treat incurable conditions worldwide.

Osteosarcoma of the tibia 6 years after tibial plateau leveling osteotomy.

A 7 yr old spayed female mastiff presented for examination of a left pelvic limb lameness of 3 mo duration. Six years previously, the dog had undergone tibial plateau leveling osteotomy (TPLO) surgery of the left pelvic limb for the treatment of cranial cruciate disease. On presentation, the dog had a painful and swollen proximal tibia. Following investigation, a diagnosis of osteosarcoma of the proximal left tibia at the site of the previous TPLO surgery was made. This is the first reported case of osteosarcoma following TPLO using an implant other than the Slocum plate.

Bite-size introduction to canine hematologic malignancies.

Hematologic malignancies are frequently diagnosed in dogs and result in a spectrum of clinical signs associated with specific disease types. The most frequently encountered hematologic tumors in dogs include lymphoma, lymphoid and myeloid leukemias, and mast cell, plasma cell, and histiocytic neoplasias. Coupled with the heterogeneous presentations of the different categories and subtypes of canine hematologic malignancies, outcomes for these tumors are also variable. Considering this, appropriate treatment options range from active surveillance to curative intent approaches harnessing surgical, chemotherapeutic, and radiation-based modalities. The underlying pathology of many of these diseases bears remarkable resemblance to that of the corresponding diagnosis made in human patients. We introduce some of the pathogenic drivers of canine hematologic cancers alongside their clinical presentations. An overview of standard-of-care therapies for each of these diseases is also provided. As comparative oncology gains recognition as a valuable setting in which to investigate the pathogenesis of neoplasia and provide powerful, clinically relevant, immunocompetent models for the evaluation of novel therapies, the number of clinicians and scientists participating in cancer research involving dogs is expected to increase. This review aims at providing an introductory overview of canine hematologic malignancies.

Case Report: Clinical and Serological Hallmarks of Cytokine Release Syndrome in a Canine B Cell Lymphoma Patient Treated With Autologous CAR-T Cells.

Background: Chimeric antigen receptor-T (CAR-T) cells have transformed the treatment of human B cell malignancies. With the advent of CAR-T therapy, specific and in some cases severe toxicities have been documented with cytokine release syndrome (CRS) being the most frequently reported. As dogs develop tumors spontaneously and in an immunocompetent setting, they provide a unique translational opportunity to further investigate the activity and toxicities associated with CAR-T therapy. Although various adoptive cellular therapy (ACT) trials have been documented and several more are ongoing in canine oncology, CRS has not been comprehensively described in canine cancer patients. Case Presentation: Here we present the clinical and serologic changes in a dog treated with autologous CAR-T for relapsed B cell lymphoma that presented with lethargy and fever 3 days following CAR-T. Multiplexed serum cytokine profiling revealed increases in key cytokines implicated in human CRS including IL-6, MCP-1, IFNγ and IL-10 at or shortly after peak CAR-T levels in vivo. Conclusion: The observations noted in this case report are consistent with CRS development following CAR-T therapy in a canine patient. The dog represents a compelling model to study the pathophysiology of CRS and pre-clinically screen novel therapeutics to prevent and treat this life-threatening condition in the setting of a complex and naturally evolved immune system.

Evaluation of the anti-tumour activity of Coriolus versicolor polysaccharopeptide (I'm-Yunity) alone or in combination with doxorubicin for canine splenic hemangiosarcoma.

Canine splenic hemangiosarcoma (HSA) is an aggressive tumour of vascular endothelium that carries a grave prognosis following standard of care treatment with surgery and doxorubicin. A previous pilot study revealed potential anti-tumour activity of I'm-Yunity polysaccharopeptide (PSP) for canine HSA. The aim of this prospective study was to assess patient outcome when treated with PSP alone or in combination with doxorubicin post-splenectomy compared to patients treated with surgery and doxorubicin that received a placebo in place of PSP. Dogs undergoing splenectomy for splenic HSA were eligible. Following splenectomy, owners were offered treatment with PSP alone or adjuvant doxorubicin chemotherapy (unblinded). Patients with owners that selected to proceed with doxorubicin chemotherapy were blindly randomized to receive placebo or PSP. Dogs were evaluated weekly for 15 weeks, then scheduled for monthly visits until death. One hundred and one dogs were included in the final analysis: 51 PSP alone, 25 doxorubicin/placebo, and 25 combination PSP/doxorubicin. On multivariate analysis, dogs treated with single agent PSP, female dogs, decreased haematocrit at diagnosis, and stage III disease were negatively significantly associated with outcome; however, an interaction between treatment group and sex was documented. Gender-specific outcomes revealed no significant difference in survival between treatment groups for male dogs, but female dogs treated with PSP alone had significantly reduced survival compared to females receiving doxorubicin/placebo (HR 0.21; p = .004). Anaemia (HR 5.28; p < .001) and stage III disease (HR 2.9; p = .014) remained negatively associated with survival when controlling for sex and treatment group. The addition of PSP to doxorubicin post-splenectomy did not improve survival in dogs with splenic HSA.

Tumor factors stimulate lysosomal degradation of tumor antigens and undermine their cross-presentation in lung cancer.

Activities of dendritic cells (DCs) that present tumor antigens are often suppressed in tumors. Here we report that this suppression is induced by tumor microenvironment-derived factors, which activate the activating transcription factor-3 (ATF3) transcription factor and downregulate cholesterol 25-hydroxylase (CH25H). Loss of CH25H in antigen presenting cells isolated from human lung tumors is associated with tumor growth and lung cancer progression. Accordingly, mice lacking CH25H in DCs exhibit an accelerated tumor growth, decreased infiltration and impaired activation of intratumoral CD8+ T cells. These mice do not establish measurable long-term immunity against malignant cells that undergo chemotherapy-induced immunogenic cell death. Mechanistically, downregulation of CH25H stimulates membrane fusion between endo-phagosomes and lysosomes, accelerates lysosomal degradation and restricts cross-presentation of tumor antigens in the intratumoral DCs. Administration of STING agonist MSA-2 reduces the lysosomal activity in DCs, restores antigen cross presentation, and increases therapeutic efficacy of PD-1 blockade against tumour challenge in a CH25H-dependent manner. These studies highlight the importance of downregulation of CH25H in DCs for tumor immune evasion and resistance to therapy.

Targeting PARP11 to avert immunosuppression and improve CAR T therapy in solid tumors.

Evasion of antitumor immunity and resistance to therapies in solid tumors are aided by an immunosuppressive tumor microenvironment (TME). We found that TME factors, such as regulatory T cells and adenosine, downregulated type I interferon receptor IFNAR1 on CD8+ cytotoxic T lymphocytes (CTLs). These events relied upon poly-ADP ribose polymerase-11 (PARP11), which was induced in intratumoral CTLs and acted as a key regulator of the immunosuppressive TME. Ablation of PARP11 prevented loss of IFNAR1, increased CTL tumoricidal activity and inhibited tumor growth in an IFNAR1-dependent manner. Accordingly, genetic or pharmacologic inactivation of PARP11 augmented the therapeutic benefits of chimeric antigen receptor T cells. Chimeric antigen receptor CTLs engineered to inactivate PARP11 demonstrated a superior efficacy against solid tumors. These findings highlight the role of PARP11 in the immunosuppressive TME and provide a proof of principle for targeting this pathway to optimize immune therapies.

Genetic re-direction of canine primary T cells for clinical trial use in pet dogs with spontaneous cancer.

Immunocompetent pet dogs develop spontaneous, human-like cancers, representing a parallel patient population for the investigation of chimeric antigen receptor (CAR) therapies. We have optimized a retrovirus-based protocol to efficiently CAR transduce primary T cells from healthy and tumor-bearing dogs. While transduction efficiencies and CAR-T expansion vary among dogs, CAR expression is typically higher and more stable compared with previous protocols, thus enabling human and comparative oncology researchers to use the dog as a pre-clinical model for human CAR-T cell research. For complete details on the use and execution of this protocol, please refer to Panjwani et al. (2020).

Response-based modification of CHOP chemotherapy for canine B-cell lymphoma.

Despite high initial response rates, a subset of dogs with B-cell lymphoma responds less robustly to CHOP-based chemotherapy and experiences shorter survival. One hundred and four dogs with nodal B-cell lymphoma were treated with a response-based CHOP (RBCHOP) protocol modified based on response to individual drugs during the first chemotherapy cycle. Dogs achieving complete (CR) or partial response (PR) at week 3, following treatment with vincristine and cyclophosphamide, received RBCHOP 1 (n = 72), a protocol sequentially rotating vincristine, cyclophosphamide, and doxorubicin. Dogs without a detectable response at week 3 that subsequently achieved CR or PR following treatment with doxorubicin received RBCHOP 2 (n = 14), in which four doses of doxorubicin were given consecutively followed by vincristine and cyclophosphamide. Dogs that failed to respond at week 3 and then to doxorubicin at week 5 assessment were offered rescue chemotherapy (RBCHOP 3, n = 18). Median progression free survival (PFS) and overall survival time (OST) were similar between RBCHOP 1 (PFS 210 days, OST 354 days) and RBCHOP 2 (PFS 220 days, OST 456 days), but significantly shorter for RBCHOP 3 (PFS 34 days, OST 80.5 days, P < 0.001). No presenting signalment nor hematologic variable differentiated patient cohort, however, dogs in RBCHOP 2 and RBCHOP 3 were more likely to have a lymphocytosis at diagnosis (P = 0.02 and 0.04, respectively). Protocol modification based on response during the first cycle resulted in similar toxicity profiles and outcomes to previously published variants of CHOP, and prognosis remained poor for dogs failing to respond during the first treatment cycle.

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.

Enhanced immunotherapeutic profile of oncolytic virus-based cancer vaccination using cyclophosphamide preconditioning.

Despite a sizeable body of research, the efficacy of therapeutic cancer vaccines remains limited when applied as sole agents. By using a prime:boost approach involving two viral cancer vaccines, we were able to generate large tumor-specific CD8+ T-cell responses in a murine model of disseminated pulmonary melanoma. Significant increases in the number and quality of circulating effector T-cells were documented when low-dose cyclophosphamide (CTX) was administered pre-vaccination to tumor-bearing but not tumor-free hosts. Interestingly, tumor-bearing mice receiving CTX and co-primed with a melanoma differentiation antigen together with an irrelevant control antigen exhibited significantly enhanced immunity against the tumor, but not the control antigen, in secondary lymphoid organs. This result highlighted an increased cancer-specific reactivity of vaccine-induced T-cell responses following CTX preconditioning. Additionally, an acute reduction of the frequency of peripheral regulatory T-cells (Tregs) was noticeable, particularly in the proliferating, presumably tumour-reactive, subset. Enhanced infiltration of lungs with multifunctional T-cells resulted in overt reduction in metastatic burden in mice pretreated with CTX. Despite doubling the median survival in comparison to untreated controls, most vaccinated mice ultimately succumbed to cancer progression. However, preconditioning of the virus-based vaccination with CTX resulted in a remarkable improvement of the therapeutic activity leading to complete remission in the majority of the animals. Collectively, these data reveal how CTX can potentiate specific cellular immunity in an antigen-restricted manner that is only observed in vaccinated tumor-bearing hosts while depleting replicating Tregs. A single low dose of CTX enhances antitumor immunity and the efficacy of this potent prime:boost platform by modulating the kinetics of the vaccine-specific responses. Clinical assessment of CTX combined with next-generation cancer vaccines is indicated.

Establishing a model system for evaluating CAR T cell therapy using dogs with spontaneous diffuse large B cell lymphoma.

Multiple rodent and primate preclinical studies have advanced CAR T cells into the clinic. However, no single model accurately reflects the challenges of effective CAR T therapy in human cancer patients. To evaluate the effectiveness of next-generation CAR T cells that aim to overcome barriers to durable tumor elimination, we developed a system to evaluate CAR T cells in pet dogs with spontaneous cancer. Here we report on this system and the results of a pilot trial using CAR T cells to treat canine diffuse large B cell lymphoma (DLBCL). We designed and manufactured CD20-targeting, second-generation canine CAR T cells for functional evaluation in vitro and in vivo using lentivectors to parallel human CAR T cell manufacturing. A first-in-species trial of five dogs with DLBCL treated with CAR T was undertaken. Canine CAR T cells functioned in an antigen-specific manner and killed CD20+ targets. Circulating CAR T cells were detectable post-infusion, however, induction of canine anti-mouse antibodies (CAMA) was associated with CAR T cell loss. Specific selection pressure on CD20+ tumors was observed following CAR T cell therapy, culminating in antigen escape and emergence of CD20-disease. Patient survival times correlated with ex vivo product expansion. Altering product manufacturing improved transduction efficiency and skewed toward a memory-like phenotype of canine CAR T cells. Manufacturing of functional canine CAR T cells using a lentivector is feasible. Comparable challenges to effective CAR T cell therapy exist, indicating their relevance in informing future human clinical trial design.

Preclinical evaluation of a MAGE-A3 vaccination utilizing the oncolytic Maraba virus currently in first-in-human trials.

Multiple immunotherapeutics have been approved for cancer patients, however advanced solid tumors are frequently refractory to treatment. We evaluated the safety and immunogenicity of a vaccination approach with multimodal oncolytic potential in non-human primates (NHP) (Macaca fascicularis). Primates received a replication-deficient adenoviral prime, boosted by the oncolytic Maraba MG1 rhabdovirus. Both vectors expressed the human MAGE-A3. No severe adverse events were observed. Boosting with MG1-MAGEA3 induced an expansion of hMAGE-A3-specific CD4+ and CD8+ T-cells with the latter peaking at remarkable levels and persisting for several months. T-cells reacting against epitopes fully conserved between simian and human MAGE-A3 were identified. Humoral immunity was demonstrated by the detection of circulating MAGE-A3 antibodies. These preclinical data establish the capacity for the Ad:MG1 vaccination to engage multiple effector immune cell populations without causing significant toxicity in outbred NHPs. Clinical investigations utilizing this program for the treatment of MAGE-A3-positive solid malignancies are underway (NCT02285816, NCT02879760).