Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years. A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1. Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism. Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers. In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs. In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated in vitro, demonstrating significantly enhanced cytokine production and proliferation of dog peripheral blood mononuclear cells. A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma. Objective antitumour responses were observed in one dog with OMM (14.3%, 1/7) and one with undifferentiated sarcoma (50.0%, 1/2) when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers.

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Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the “exhausted” status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1–expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-γ production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed.

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Spontaneous cancers are common diseases in dogs. Among these, some malignant cancers such as oral melanoma, osteosarcoma, hemangiosarcoma, and mast cell tumor are often recognized as clinical problems because, despite their high frequencies, current treatments for these cancers may not always achieve satisfying outcomes. The absence of effective systemic therapies against these cancers leads researchers to investigate novel therapeutic modalities, including immunotherapy. Programmed death 1 (PD-1) is a costimulatory receptor with immunosuppressive function. When it binds its ligands, PD-ligand 1 (PD-L1) or PD-L2, PD-1 on T cells negatively regulates activating signals from the T cell receptor, resulting in the inhibition of the effector function of cytotoxic T lymphocytes. Aberrant PD-L1 expression has been reported in many human cancers and is considered an immune escape mechanism for cancers. In clinical trials, anti-PD-1 or anti-PD-L1 antibodies induced tumor regression for several malignancies, including advanced melanoma, non-small cell lung carcinoma, and renal cell carcinoma. In this study, to assess the potential of the PD-1/PD-L1 axis as a novel therapeutic target for canine cancer immunotherapy, immunohistochemical analysis of PD-L1 expression in various malignant cancers of dogs was performed. Here, we show that dog oral melanoma, osteosarcoma, hemangiosarcoma, mast cell tumor, mammary adenocarcinoma, and prostate adenocarcinoma expressed PD-L1, whereas some other types of cancer did not. In addition, PD-1 was highly expressed on tumor-infiltrating lymphocytes obtained from oral melanoma, showing that lymphocytes in this cancer type might have been functionally exhausted. These results strongly encourage the clinical application of PD-1/PD-L1 inhibitors as novel therapeutic agents against these cancers in dogs.

Abstract-Phospholipase C (PLC) ⑀ is a recently identified enzyme regulated by a wide range of molecules including Ras family small GTPases, Rho A, G␣ 12/13 , and G␤␥ with primary sites of expression in the heart and lung. In a screen for human signal transduction genes altered during heart failure, we found that PLC⑀ mRNA is upregulated. Two murine models of cardiac hypertrophy confirmed upregulation of PLC⑀ protein expression or PLC⑀ RNA. To identify a role for PLC⑀ in cardiac function and pathology, a PLC⑀-deficient mouse strain was created. Echocardiography indicated PLC⑀ Ϫ/Ϫ mice had decreased cardiac function, and direct measurements of left ventricular contraction demonstrated that PLC⑀ Ϫ/Ϫ mice had a decreased contractile response to acute isoproterenol administration. Cardiac myocytes isolated from PLC⑀ Ϫ/Ϫ mice had decreased ␤-adrenergic receptor (␤AR)-dependent increases in Ca 2ϩ transient amplitudes, likely accounting for the contractile deficiency in vivo. This defect appears to be independent from the ability of the ␤AR system to produce cAMP and regulation of sarcoplasmic reticulum Ca 2ϩ pool size. To address the significance of these functional deficits to cardiac pathology, PLC⑀ Ϫ/Ϫ mice were subjected to a chronic isoproterenol model of hypertrophic stress. PLC⑀ Ϫ/Ϫ mice were more susceptible than wild-type littermates to development of hypertrophy than wild-type littermates. Together, these data suggest a novel PLC-dependent component of ␤AR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve and loss of PLC⑀ signaling sensitizes the heart to development of hypertrophy in response to chronic cardiac stress. Key Words: phospholipase C Ⅲ ␤-adrenergic receptor Ⅲ heart failure Ⅲ contractility A gonist regulation of intracellular calcium and protein kinase C (PKC) signaling through activation of phospholipase C (PLC) modulates a wide range of physiological responses. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to generate inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol, which increase cytosolic calcium concentration and activate PKC, respectively. Five PLC families have been identified as ␤, ␥, ␦, , and ⑀. All contain X and Y domains that form the catalytic core for PIP 2 -PLC hydrolysis activity, whereas each isoform has unique domains involved in physiological regulation by distinct signaling pathways. PLC␤ and -␥ isoforms are well-studied enzymes that are regulated by G protein-coupled receptors and receptor tyrosine kinases, respectively. 1,2 PLC⑀ is a novel PLC isoform that has been shown to be regulated by Ras, 3 Rap, [3][4][5] RhoA, 5,6 RalA, 5 TC21, 5 Rac, 5 G 12/13 , 5,7 and G␤␥ 8 in transfected cells. PLC⑀ is also a unique bifunctional enzyme that, in addition to PLC catalytic activity, has an N-terminal domain with homology to the Ras guanine nucleotide exchange factor (GEF) CDC25 and has GEF activity toward Rap. 9 A tissue with significant levels of PLC⑀ expression is the heart. Roles for specific PLC isoforms in cardiac func...

Tumor necrosis factor-alpha exacerbates myocardial I/R injury at an early stage of reperfusion by activating NF-kappaB, thereby inducing chemokines and adhesion molecules and facilitating leukocyte infiltration.

Immunotherapy targeting programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) represents promising treatments for human cancers. Our previous studies demonstrated PD-L1 overexpression in some canine cancers, and suggested the therapeutic potential of a canine chimeric anti-PD-L1 monoclonal antibody (c4G12). However, such evidence is scarce, limiting the clinical application in dogs. In the present report, canine PD-L1 expression was assessed in various cancer types, using a new anti-PD-L1 mAb, 6C11-3A11, and the safety and efficacy of c4G12 were explored in 29 dogs with pulmonary metastatic oral malignant melanoma (OMM). PD-L1 expression was detected in most canine malignant cancers including OMM, and survival was significantly longer in the c4G12 treatment group (median 143 days) when compared to a historical control group (n = 15, median 54 days). In dogs with measurable disease (n = 13), one dog (7.7%) experienced a complete response. Treatment-related adverse events of any grade were observed in 15 dogs (51.7%). Here we show that PD-L1 is a promising target for cancer immunotherapy in dogs, and dogs could be a useful large animal model for human cancer research.