Development of an in vitro assay for screening programmed death receptor-1/programmed cell death ligand 1 monoclonal antibody therapy in dogs.

Immunomodulatory antibody drugs that modulate the function of immune checkpoint molecules, such as programmed death receptor-1 (PD-1) and programmed cell death ligand 1 (PD-L1), have been established as new cancer treatments in human medicine. In recent years, there have also been reports on antibodies that inhibit immune checkpoint molecules in dogs, and clinical trials using such antibodies for canine cancer have been gradually increasing in number. Because inhibitory antibodies restore T-cell function by inhibiting the binding of PD-1 on T cells and its ligand PD-L1, the quality of antibody function has been evaluated using activated T cells or peripheral blood mononuclear cells isolated from healthy dogs; however, the assays and dogs used significantly vary. Therefore, in the present study, we developed a reporter gene assay using reporter cells (Jurkat/NFATluc/cPD1) and effector cells (CTAC/OKT3/cPDL1). Jurkat/NFATluc/cPD1 were generated by introducing both of the NFAT-responsive luciferase gene as a marker of T-cell signaling and canine PD-1, into a human T lymphoid cell line, Jurkat. CTAC/OKT3/cPDL1 were generated by introducing single-chain FV (scFV) of anti-human CD3 antibody (OKT3) and canine PD-L1 into a canine thyroid carcinoma cell line, CTAC. Ligation of PD-1 on Jurkat/NFATluc/cPD1 via binding of PD-L1 on CTAC/OKT3/cPDL1 suppressed NFAT luciferase activity induced by CD3 ligation by scFV of OKT3. The addition of anti-canine PD-1 and PD-L1 antibodies, both of which were previously developed in our laboratory, restored this suppression with high sensitivity, although the anti-human PD-L1 antibody atezolizumab induced a very weak restoration. This assay is an useful method for functionally evaluating the inhibition of canine PD-1 and PD-L1 binding.

Proof-of-concept study of the caninized anti-canine programmed death 1 antibody in dogs with advanced non-oral malignant melanoma solid tumors.

BACKGROUND: The anti-programmed death 1 (PD-1) antibody has led to durable clinical responses in a wide variety of human tumors. We have previously developed the caninized anti-canine PD-1 antibody (ca-4F12-E6) and evaluated its therapeutic properties in dogs with advance-staged oral malignant melanoma (OMM), however, their therapeutic effects on other types of canine tumors remain unclear. OBJECTIVE: The present clinical study was carried out to evaluate the safety profile and clinical efficacy of ca-4F12-E6 in dogs with advanced solid tumors except for OMM. METHODS: Thirty-eight dogs with non-OMM solid tumors were enrolled prospectively and treated with ca-4F12-E6 at 3 mg/kg every 2 weeks of each 10-week treatment cycle. Adverse events (AEs) and treatment efficacy were graded based on the criteria established by the Veterinary Cooperative Oncology Group. RESULTS: One dog was withdrawn, and thirty-seven dogs were evaluated for the safety and efficacy of ca-4F12-E6. Treatment-related AEs of any grade occurred in 13 out of 37 cases (35.1%). Two dogs with sterile nodular panniculitis and one with myasthenia gravis and hypothyroidism were suspected of immune-related AEs. In 30 out of 37 dogs that had target tumor lesions, the overall response and clinical benefit rates were 6.9% and 27.6%, respectively. The median progression-free survival and overall survival time were 70 days and 215 days, respectively. CONCLUSIONS: The present study demonstrated that ca-4F12-E6 was well-tolerated in non-OMM dogs, with a small number of cases showing objective responses. This provides evidence supporting large-scale clinical trials of anti-PD-1 antibody therapy in dogs.

Seroprevalence of Dermatophagoides farinae-specific IgE in dogs with atopic dermatitis in São Paulo, Brazil.

Canine atopic dermatitis (CAD) is a chronic, inflammatory, and pruritic disease of the skin resulting from the loss of the epidermal barrier, sensitization, and exacerbated production of IgE antibodies mainly directed against environmental allergens, especially to house dust mites. To select specific allergen immunotherapies with high efficacy, there are necessary studies with house dust mite allergens to improve both serological and intradermal tests. Therefore, the objective of this study was to evaluate the seroprevalence of IgE against Der f 2, Zen 1, and crude Dermatophagoides farinae allergens in dogs with AD in the State of São Paulo, Brazil. The sera of 85 dogs with clinically confirmed atopic dermatitis from the State of São Paulo (Brazil) was collected. In addition, an indirect ELISA test was conducted to detect allergen-specific serum IgE. IgE seropositivity was observed in 97.5% of the dogs for Der f 2, 95.0% for Zen 1, and 92.5% for the crude mite allergens. Due to this high prevalence of IgE specific to these allergens, we suggest that Der f 2 and Zen 1 can be considered major allergens for dogs in the State of São Paulo.

Antimicrobial resistance and genotyping of Pseudomonas aeruginosa isolated from the ear canals of dogs in Japan.

The strong bond between dogs and their owners creates a close association that could result in the transfer of antibiotic-resistant bacteria from canines to humans, potentially leading to the spread of antimicrobial resistance genes. Pseudomonas aeruginosa, a common causative agent of persistent ear infections in dogs, is often resistant to multiple antibiotics. Assessing the antimicrobial resistance profile and genotype of P. aeruginosa is crucial for the appropriate use of veterinary pharmaceuticals. However, in recent years, few studies have been conducted on this bacterium in Japan. We determined the antimicrobial resistance profile and genotype of P. aeruginosa isolated from the ear canal of dogs in Japan in 2020. Analysis of antimicrobial resistance using disk diffusion tests indicated a high frequency of resistance to most antimicrobial agents. Particularly, 29 isolates from the ear canals of the 29 affected dogs (100%) were resistant to cefovecin, cefpodoxime, and florfenicol; however, they were susceptible to cefepime and piperacillin/tazobactam. Only 3.4, 10.3, and 10.3% of the isolates were resistant to ceftazidime, tobramycin, and gentamicin, respectively. Furthermore, upon analyzing the population structure using multilocus sequence typing, a considerably large clonal complex was not observed in the tested isolates. Three isolates, namely ST3881, ST1646, and ST532, were clonally related to the clinically isolated sequence types in Japan (such as ST1831, ST1413, ST1812, and ST1849), which is indicative of dog-to-human transmission. Considering the variation in antibiotic resistance compared to that reported by previous studies and the potential risk of dog-to-human transmission, we believe that the survey for antimicrobial resistance profile and population structure should be continued regularly. However, the prevalence of multidrug-resistant P. aeruginosa in dogs in Japan is not a crisis.

Long-term survival of dogs with stage 4 oral malignant melanoma treated with anti-canine PD-1 therapeutic antibody: A follow-up case report.

A monoclonal antibody targeting programmed cell death-1 (PD-1) is one of the most promising treatments for human cancers. Clinical studies in humans demonstrated that the anti-PD-1 antibody provides a long-lasting tumour response. Previously, we established an anti-canine PD-1 therapeutic antibody (ca-4F12-E6), and the pilot clinical study demonstrated that the antibody was effective in dogs with oral malignant melanoma (OMM). However, two OMM cases were still undergoing treatment when the pilot study was published. Here, we describe the long-term follow-up of those two cases. Although both cases showed long-term survival with complete response (CR), the tumour response differed; the effect onset was slow in one case and a durable response was observed in the second case even after treatment discontinuation. Secondary malignant tumours occurred during treatment in both cases. This follow-up study revealed that ca-4F12-E6 maintains CR in dogs for more than 1 year. In addition, the pattern of tumour response was unique compared to conventional therapy. These results indicate that new evaluation criteria for tumour response may be necessary for immunotherapy in veterinary medicine. Long-term follow-up is necessary regardless of the short-term treatment responsiveness.

Development of a monoclonal antibody for the detection of anti-canine CD20 chimeric antigen receptor expression on canine CD20 chimeric antigen receptor-transduced T cells.

Chimeric antigen receptor (CAR) CAR-T cell therapy targeting CD20 can be a novel adoptive cell therapy for canine patients with B-cell malignancy. After injection of the CAR-T cells in vivo, monitoring circulating CAR-T cells is essential to prove in vivo persistence of CAR-T cells. In this study, we developed a novel monoclonal antibody against canine CD20 CAR, whose single-chain variable fragment was derived from the our previously reported anti-canine CD20 therapeutic antibody. Furthermore, we proved that this monoclonal antibody can detect therapeutic anti-canine CD20 chimeric antibody in the serum from healthy beagle dogs injected with the therapeutic antibody for safety study. This monoclonal antibody is a useful tool for monitoring both canine CD20-CAR-T cells and anti-canine CD20 therapeutic antibody for canine lymphoma.

Development of a cell line-based assay to measure the antibody-dependent cellular cytotoxicity of a canine therapeutic antibody.

Antibody drugs are one of the most important therapeutic modalities in cancer treatment. Antibody drugs for canine cytotoxicity have been developed, and the most important way to evaluate the function of antibody drugs in vitro is to measure the antibody-dependent cellular cytotoxicity (ADCC) activity. However, a good method for measuring the ADCC activity of antibody drugs for dogs has not yet been developed. This study generated a cell line of NK-92 cells, a human natural killer cell line, transfected with canine CD16 and γ-chain as effector cells. A20 cells expressing canine CD20 were used as target cells, and a system for ADCC activity was established using an anti-dog CD20 antibody. The assay generated very consistent results. This is an applicable method for measuring canine ADCC activity and can be used for various antigen-antibody combinations to develop canine antibody drugs with cytotoxic activity in the future.

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.

Serum canine thymus and activation-regulated chemokine (TARC/CCL17) concentrations correlate with disease severity and therapeutic responses in dogs with atopic dermatitis.

BACKGROUND: Thymus and activation-regulated chemokine (TARC/CCL17) has been implicated in the pathogenesis of canine atopic dermatitis (cAD). Serum TARC concentrations are a reliable biomarker for human atopic dermatitis; however, their potential as a biomarker for cAD has not been investigated. HYPOTHESIS/OBJECTIVES: To investigate whether serum TARC concentrations correlate with disease severity and therapeutic responses for cAD. ANIMALS: Thirty-nine dogs with cAD and 42 healthy dogs were recruited. METHODS AND MATERIALS: Serum TARC concentrations in dogs with cAD and healthy dogs were measured by sandwich ELISA with anti-canine TARC antibodies. The clinical severity of cAD was scored using the validated Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04). Serum TARC concentrations were compared between dogs with cAD and healthy controls, and their relationship with CADESI-04 was examined. Serum TARC concentrations also were measured in 20 dogs with cAD treated with prednisolone or oclacitinib for four weeks. RESULTS: Serum TARC concentrations were significantly higher in dogs with cAD than in healthy dogs (P < 0.001). In dogs with cAD, serum TARC concentrations correlated with CADESI-04 scores (ρ = 0.457, P < 0.01). Furthermore, serum TARC concentrations significantly decreased in treated dogs with the attenuation of clinical signs (P < 0.001). Changes in serum TARC concentrations before and after treatment correlated with those in CADESI-04 scores (ρ = 0.746, P < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: Serum TARC concentrations have potential as a clinical and research tool for the objective evaluation of disease severity and therapeutic responses for cAD.

Generation of a canine anti-canine CD20 antibody for canine lymphoma treatment.

Lymphoma is the most common hematological cancer in dogs. Canine diffuse large B cell lymphoma shows a relatively good response to treatment with multi-agent cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy; however, the 2-year survival rate is as low as 20%. For human B cell type lymphoma, the anti-CD20 chimeric antibody, rituximab, was developed two decades ago. The combination of rituximab and CHOP chemotherapy was highly successful in improving patient prognosis. However, no anti-canine CD20 antibody is available for the treatment of canine lymphoma. During this study, a rat anti-canine CD20 monoclonal antibody was established. We also generated a rat-canine chimeric antibody against canine CD20 designed for clinical application. This chimeric antibody (4E1-7-B) showed in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the canine B cell lymphoma cell line CLBL-1. Moreover, to obtain stronger ADCC activity, a defucosylated 4E1-7-B antibody (4E1-7-B_f) was also generated, and it showed tenfold stronger ADCC activity compared with 4E1-7-B. 4E1-7-B_f as well as 4E1-7-B suppressed the growth of CLBL-1 tumors in an immunodeficient xenotransplant mouse model. Finally, a single administration of 4E1-7-B_f induced considerable peripheral B cell depletion in healthy beagles. Thus, 4E1-7-B_f is a good antibody drug candidate for canine B cell type lymphoma.

Establishment of rat anti-canine DEP domain containing 1B (DEPDC1B) monoclonal antibodies.

DEP domain-containing 1B (DEPDC1B) is involved in the regulation of cell de-adhesion and actin cytoskeleton activity during the G2/M transition of the cell cycle, and its overexpression has been proven to be associated with cancer progression in several human cancers. Canine DEPDC1B was identified as a gene that was overexpressed in canine lymphoma tissues in our previous study. However, in dogs, the protein expression of DEPDC1B remains to be determined due to the lack of a specific monoclonal antibody. Here, we developed rat monoclonal antibodies against canine DEPDC1B and characterized their applicability for immunodetection assays. Our findings demonstrated that these antibodies are functional and can be important tools to investigate the precise role of DEPDC1B in canine tumors.

Therapeutic Potential of an Endolysin Derived from Kayvirus S25-3 for Staphylococcal Impetigo.

Impetigo is a contagious skin infection predominantly caused by Staphylococcus aureus. Decontamination of S. aureus from the skin is becoming more difficult because of the emergence of antibiotic-resistant strains. Bacteriophage endolysins are less likely to invoke resistance and can eliminate the target bacteria without disturbance of the normal microflora. In this study, we investigated the therapeutic potential of a recombinant endolysin derived from kayvirus S25-3 against staphylococcal impetigo in an experimental setting. First, the recombinant S25-3 endolysin required an incubation period of over 15 minutes to exhibit efficient bactericidal effects against S. aureus. Second, topical application of the recombinant S25-3 endolysin decreased the number of intraepidermal staphylococci and the size of pustules in an experimental mouse model of impetigo. Third, treatment with the recombinant S25-3 endolysin increased the diversity of the skin microbiota in the same mice. Finally, we revealed the genus-specific bacteriolytic effect of recombinant S25-3 endolysin against staphylococci, particularly S. aureus, among human skin commensal bacteria. Therefore, topical treatment with recombinant S25-3 endolysin can be a promising disease management procedure for staphylococcal impetigo by efficient bacteriolysis of S. aureus while improving the cutaneous bacterial microflora.

The detection of house dust mite Dermatophagoides farinae, Der f 2 and Zen-1 allergen-specific immunoglobulin E antibodies in dogs with atopic Dermatitis in Malaysia.

Canine atopic dermatitis (AD) is a chronic, inflammatory and pruritic allergic skin disease in dogs. House dust mites such as Dermatophagoides farinae are one of the known causative agents for the induction of canine AD worldwide. D. farinae protein Der f 2 is known as an important allergen involved in canine AD and recently, Zen-1 has also been identified as an allergenic protein. There is limited information on the prevalence and role of allergen sensitization to crude D. farinae extract (CDF), Der f 2 and Zen-1 among dogs diagnosed with AD in Malaysia. The aim of this study was to determine the proportion of CDF-, Der f 2- and Zen-1-specific reactive sera among dogs diagnosed with AD in Malaysia using an enzyme-linked immunosorbent assay (ELISA). Serum samples were collected from dogs diagnosed with AD from several veterinary clinics in Malaysia. The canine case records were retrieved and information on signalment, dermatological and non-dermatological histories, clinical presentation, food allergies, and exclusion of ectoparasitic, microbial and fungal skin infections were obtained through a survey form. All serum samples were evaluated to quantify the CDF-, Der f 2- and Zen-1-specific immunoglobulin E (IgE) levels. A total of 24.6%, 48.4% and 29.8% of dogs diagnosed with AD were positive for CDF-, Der f 2- and Zen-1-specific IgE, respectively. These results suggest that CDF-, Der f 2- and Zen-1 are important allergens that can contribute to AD in dogs in Malaysia, and serological testing can be performed to provide additional treatment options involving specific immunotherapies.

B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that B cell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

Th17 cells increase during maturation in peripheral blood of healthy dogs.

Recent studies have indicated that T helper 17 (Th17) cells are involved in the pathogenesis of various inflammatory diseases in dogs. However, age-related changes in canine Th17 cells have not yet been investigated. In the present study, the proportion of Th17 cells was examined in the peripheral blood mononuclear cells (PBMCs) of healthy dogs at various ages: Group 1 (n = 16; less than 1 year of age), Group 2 (n = 25; 1-5 years), and Group 3 (n = 19; 6-9 years), using flow cytometry and an anti-human interleukin (IL)-17A monoclonal antibody that reacts with canine IL-17A. The proportion of circulating Th17 cells positively correlated with age. The age-related differences were observed in the proportion of Th17 cells among Group 1 (mean ± SD: 1.52 ± 1.18%), Group 2 (mean ± SD: 3.81 ± 1.94%) and Group 3 (mean ± SD: 7.49 ± 2.54%). Our results suggest that age-related changes in Th17 cells need to be considered in future research on Th17-related diseases in dogs.

Localization of a mutant SOD1 protein in E40K-heterozygous dogs: Implications for non-cell-autonomous pathogenesis of degenerative myelopathy.

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disorder. Dogs with typical clinical signs carry homozygous mutations in the superoxide dismutase 1 (SOD1) gene; therefore, DM is regarded as a naturally-occurring model of amyotrophic lateral sclerosis (ALS). Despite the presence of a toxic mutant protein, E40K-SOD1 heterozygotes rarely develop clinical signs. Therefore, E40K-heterozygotes may provide insights into the subclinical and early phase of mutant SOD1-related pathology. In order to identify the distribution of mutant SOD1 in the spinal cords of E40K-heterozygotes, we developed a monoclonal antibody 16G9 that reacts to the mutant E40K-SOD1 protein. We found that the spinal cords of E40K-heterozygotes display white matter degeneration, the severity of which was markedly less than that in E40K-homozygotes. In E40K-heterozygotes, 16G9-reactive SOD1 accumulated predominantly in reactive astrocytes, while spinal neurons remained almost completely free of this form of SOD1 proteins. In contrast, all symptomatic E40K-homozygotes contained 16G9-reactive SOD1 in their spinal neurons and reactive astrocytes. These results suggest that mutant SOD1 proteins accumulate in reactive astrocytes during the early phase of DM pathology, which may contribute to subclinical neurodegeneration. The early involvement of reactive astrocytes in the pathogenesis of DM is strongly suspected and warrants further investigations in the context of non-cell autonomous neuronal death, as proposed for ALS.

Current status of synthetic hemozoin adjuvant: A preliminary safety evaluation.

Although adjuvants are a "must-have" component of successful vaccines, there are very few adjuvants licensed for use in humans, there is therefore an urgent need to develop new and safer adjuvants. Synthetic hemozoin (sHZ), a chemical analog of hemozoin which is produced by the malaria parasite, exhibits a potent adjuvant effect which enhances antigen-specific immune responses to vaccines. The potency of sHZ adjuvanticity is not limited to malaria specific vaccines, it has also been demonstrated to be effective in influenza and dog allergy models. While the synthesis of uniformly sized sHZ with consistent characteristics has proven difficult, we have recently successfully optimized the manufacture of sHZ product with an optimal adjuvant effect. Here, we summarize recent developments on the adjuvant properties of optimized sHZ adjuvant, including its good laboratory practice (GLP) non-clinical safety profile in animals. These studies ensure the safety of optimized sHZ product to be readily used as vaccine adjuvant beforehand in veterinary medicine.

Infection studies of nontarget mammalian cell lines with Bombyx mori macula-like virus.

Bombyx mori-derived cell lines are generally used for Bombyx mori nucleopolyhedrovirus (BmNPV)-based baculovirus expression vector system (BEVS). However, almost all of the B. mori-derived cell lines are persistently infected with Bombyx mori macula-like virus (BmMLV). In this study, nontarget mammalian cell lines were exposed to BmMLV, and their susceptibility was investigated. Real-time PCR showed that viral RNA in virus-inoculated nine mammalian cell lines decreased sharply at 7 days postinfection. Also, there was no significant effect on cell viability of mammalian cells after inoculation with BmMLV. These findings indicate that mammalian cell lines used in this study are not permissive to BmMLV, and BmMLV contamination might not affect the safety aspect of BmNPV-based BEVS.

IgE reactivity to a Cry j 3, an allergen of Japanese cedar (Cryptomeria japonica) pollen in dogs with canine atopic dermatitis.

The present study investigated IgE reactivity to a new Cryptomeria japonica pollen allergen (Cry j 3) in dogs with atopic dermatitis by using a fluorometric ELISA. Serum samples from 15 dogs that showed IgE sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to each allergen, individually. All 15 dogs had anti-Cry j 1 IgE, 6 (40%) had anti-Cry j 2 IgE, and 11 (73%) had anti-Cry j 3 IgE. Further, we found that these anti-Cry j 3 IgE reacted to Cry j 3 with immunoblotting analysis. These findings indicate that Cry j 3 may be a major allergen in dogs.

Measurement for canine IgE using canine recombinant high affinity IgE receptor α chain (FcεRIα).

To detect allergen-specific IgE in dogs with allergic diseases, we developed a recombinant canine high affinity IgE receptor α chain (FcεRIα)-based IgE detection system. Using the recombinant protein of canine FcεRIα expressed by an Escherichia coli expression system, we could detect house dust mite (Dermatophagoides farinae) allergen-specific IgE in sera from dogs naturally and experimentally sensitized to this allergen with ELISA and western blotting. The IgE binding activity of recombinant canine FcεRIα on ELISA was impaired by heat treatment of these sera. The specificity of this recombinant canine FcεRIα-based IgE detection system was confirmed by inhibition assays with canine IgE. The recombinant canine FcεRIα-based IgE detection system established in this study offers an alternative tool to measure allergen-specific IgE in dogs.