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Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, however, the response rates in patients with cancer are limited (∼20%–40%). To improve the success of immune checkpoint blockade therapy, relevant preclinical animal models are essential for the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs can generate knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies targeting canine immune checkpoint molecules such as canine PD-L1 (cPD-L1) have not been commercially available. Here, we developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties in multiple assays. We also evaluated the therapeutic efficacy of cPD-L1 antibodies in our unique caninized PD-L1 mice. Together, these in vitro and in vivo data, which include an initial safety profile in laboratory dogs, support development of this cPD-L1 antibody as an immune checkpoint inhibitor for studies in dogs with naturally occurring cancer for translational research. Our new therapeutic antibody and caninized PD-L1 mouse model will be essential translational research tools in raising the success rate of immunotherapy in both dogs and humans. Significance: Our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.

The quality attributes of the purified anti-cPD-L1, 1210E4 (12C) chimeric antibody. A, Canine PD-L1, 12C10E4 chimeric antibody expression construct, pTRIOZ-cIgG2-cPD-L1 12C10E4. B, SDS-PAGE analysis of 12C chimeric antibody purity under nonreducing and reducing (2-mercaptoethanol) conditions. HC, heavy chain; LC, light chain; SM, protein size marker. C,IEF analysis of 12C chimeric antibody. Standard, pI standard. D, Peptide mapping analysis. The peptide mapping of cPD-L1 12C chimeric antibody. The 12C chimeric antibody was enzymatically digested with trypsin on S-trap micro columns from Protifi after reduction and alkylation. Peptides were then separated and analyzed by RP-LC/MS-MS. The resultant mass spectrometric data were analyzed using the PEAK PTM workflow in the PEAKS X PRO Studio 10.6 software package from Bioinformatics solutions Inc. to map the detected MS1 and MS2 ions to the amino acid sequence of antibody. The sequence coverage of heavy and light chains was 100% (453 of 453 amino acids) and 98.2 (223 of 227 amino acids), respectively. E,SEC analysis of 12C chimeric antibody. Standard, SEC standard. F, MALDI-MS profiling of permethylated N-glycans released from PNGase F-treated 12C chimeric antibody. The masses of indicated glycan species represent the [M + Na+] values.

Working flow chart for production and validation of the cPD-L1 antibody. A, Immunization of antigen, cPD-L1 protein. B, Establishment of hybridomas (over 2,000 clones). C, Evaluation of the cPD-L1 specific antibody by the live cell–based antibody binding assay. D, Therapeutic antibody selection by cPD-L1/cPD-1 blockade assay. E,In vivo validation of therapeutic efficacy of cPD-L1 antibodies in the caninized PD-L1 mice. F, Production of the cPD-L1 chimeric antibody. G, Validation of the cPD-L1 chimeric antibody. H, Initial safety profile and PK analysis. PK, pharmacokinetic.

Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, however, the response rates in patients with cancer are limited (∼20%–40%). To improve the success of immune checkpoint blockade therapy, relevant preclinical animal models are essential for the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs can generate knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies targeting canine immune checkpoint molecules such as canine PD-L1 (cPD-L1) have not been commercially available. Here, we developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties in multiple assays. We also evaluated the therapeutic efficacy of cPD-L1 antibodies in our unique caninized PD-L1 mice. Together, these in vitro and in vivo data, which include an initial safety profile in laboratory dogs, support development of this cPD-L1 antibody as an immune checkpoint inhibitor for studies in dogs with naturally occurring cancer for translational research. Our new therapeutic antibody and caninized PD-L1 mouse model will be essential translational research tools in raising the success rate of immunotherapy in both dogs and humans.Significance:Our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.

Evaluation of the caninized cPD-L1 chimeric antibody. A, 12C antibody binding on the BT549cPD-L1 cells. B, Flow cytometric analysis of the 12C chimeric antibody on the BT549cPD-L1 cells. cIgG serves as a negative control. C, 12C chimeric antibody binding to cPD-L1 and cPD-L2. D, Binding affinity (KD) analysis of 12C chimeric antibody by Octet. E, EC50 of 12C chimeric antibody, 12C10E4. EC50 = 0.419 μg/mL. The bound cPD-1 protein was quantified by measuring green fluorescence at the IncuCyte S3. F and G, Canine IO Panel (NanoString) analyses were used to query changes in gene expression upon activation of cPBMCs from three healthy pet dogs. The RNA from the resting and activated PBMCs was used for NanoString work. The Canine IO panel was used to query the changes in approximately 700 genes. Groupwise analyses were conducted using “Rosalind.” There were 65 genes that were differentially expressed when comparing control PBMCs with activated PBMCs (P < 0.05, FC > 1.5) including 30 upregulated and 35 downregulated genes. In the heatmap, each column consists of data from one sample. IFNγ (H) and TNFα (I) concentrations were analyzed in the activated canine PBMCs. J and K, Flow cytometric analysis of cPD-L1 protein expression on the K9TCC or the nuclear-restricted RFP-expressing K9TCC (K9TCCnRFP) cells using the 12C chimeric antibody. The endogenous PD-L1 expression was stimulated by 50 ng/mL canine IFNγ for 12 hours. cIgG served as a negative control. L, The quantitative RT-PCR analysis of cPD-L1 (CD274) mRNA expression in the K9TCC or K9TCCnRFP cells. M, The 12C chimeric antibody enhances the tumor cell killing. Canine bladder cancer, K9TCC cells were cocultured with cPBMCs that were activated with CD3 antibody (100 ng/mL) and IL2 (10 ng/mL) at a ratio of 1 tumor cell: 15 cPBMCs. The live tumor cell count at 72 hours is shown in the bar graph. N, IFNγ concentrations were analyzed in the medium from the coculture of the K9TCC cells and activated cPBMCs with/without the 12C chimeric antibody treatment.

The high-throughput screening of therapeutic antibodies. A, Schematic diagram of the cPD-L1 antibody binding assay. BT549 cells expressing cPD-L1 were seeded on 96-well or 384-well plates. cPD-L1 antibodies (from hybridomas) and Alexa Fluor 488–conjugated anti-mouse IgG Fc-specific secondary antibody were added, and then green fluorescence signal was measured to quantify the amount of bound PD-L1 antibody by IncuCyte S3. B, A representative result of the cPD-L1 antibody binding assay. Kinetic graphs from each well of a 96-well plate showing quantitative binding of cPD-L1 antibodies on BT549 cells expressing cPD-L1 at 6-hour time intervals. The positive clones are highlighted in red (A2, I6, and I11). C, Representative images (at 18 hours) of cPD-L1 antibody binding. Green fluorescent merged images of cPD-L1–expressing cells are shown. D, Schematic diagram of the cPD-L1/cPD-1 blockade assay. BT549 cells expressing cPD-L1 were seeded on 96-well or 384-well plates. cPD-1-human IgG Fc (hFc) protein, Alexa Fluor 488–conjugated anti-human IgG Fc-specific secondary antibody and/or cPD-L1 antibody were added, and then green fluorescence signal was measured to quantify the amount of bound PD-1 protein by IncuCyte S3. E, A representative result of the cPD-L1/cPD-1 blockade assay. Kinetic graphs from each well of a 96-well plate showing quantitative binding of cPD-1 protein on BT549 cells expressing cPD-L1 at 3-hour intervals after the addition of cPD-L1 antibodies. The positive clones that blocked the interaction of cPD-L1/cPD-1 proteins are highlighted in red (A4 and B8). F, Representative images (at 18 hours) of the cPD-L1/cPD-1 blockade. Green fluorescent merged images of cPD-L1–expressing cells are shown. Note the lack of fluorescence due to the antibody binding to PD-L1 and blocking the interaction with cPD-1.

cPD-L1 antibodies enhance antitumor immunity in the caninized PD-L1 syngeneic mouse model. A, Knock-in strategy of the caninized PD-L1 mice (c57BL/c background). B, Validation of cPD-L1 protein expression in the MB49cPD-L1 cells. Flow cytometric analysis of membrane located mPD-L1 and cPD-L1 protein in MB49 cells expressing cPD-L1 (MB49cPD-L1) or MB49 parental cells. C, Immunofluorescence staining and protein expression pattern of mPD-L1 and cPD-L1 in MB49 or MB49cPD-L1 tumor masses from the caninized PD-L1 mice. DAPI, nuclear counterstaining. Scale bar, 100 μm. D, Interaction of cPD-1 or mPD-1 protein with cPD-L1 or mPD-L1 protein with or without cPD-L1 antibody, 12C. His-tagged canine or mPD-L1 protein was immobilized on the Ni-NTA 96-well plate, and HRP-conjugated anti-human IgG Fc-specific secondary with mPD-1-hFc or cPD-1-hFc protein was added. OD450 was measured to quantify the amount of bound PD-1 protein. E, Binding of cPD-L1 antibodies, 12C and 3C, with human PD-L1 (hPD-L1), mPD-L1, and cPD-L1 proteins. His-tagged human PD-L1, mPD-L1, or cPD-L1 protein was immobilized on the Ni-NTA 96-well plate, and anti-cPD-L1 antibodies, 12C or 3C, with HRP-conjugated anti-canine IgG-specific secondary was added. OD450 was measured to quantify the amount of bound PD-L1 antibodies. Ab, antibody. F, Tumor growth of MB49cPD-L1 in the caninized PD-L1 mice treated with cPD-L1 antibody, 12C or 3C. The IgG isotype of 12C and 3C antibodies is mouse IgG1 which is equivalent to human IgG4. Tumors were measured at the indicated timepoints (n = 8 per group). At the endpoint, the tumors were dissected. G–I, Immunofluorescence staining, and protein expression pattern of CD8 and granzyme B in MB49 tumor masses from IgG-, 12C-, or 3C-treated mice. DAPI, nuclear counterstaining. Scale bar, 100 μm. Representative images of immunostaining of CD8 and granzyme B in the MB49 tumor mass (G). CD8 (H) and granzyme B (I) were quantified using Gen5 software (BioTek). n = 10. Treatment with the PD-L1 antibody did not affect kidney function (serum creatinine; J) or liver enzyme activity (ALT; K), measured in blood collected at the end of the experiment. ALT, alanine aminotransferase.

Pharmacokinetic analysis of 12C chimeric antibody in laboratory dogs. A, Schematic diagram of ELISA for pharamacokinetic analysis. The concentration of the 12C chimeric antibody was measured in the 2 mg/kg (B) or 5 mg/kg (C) 12C antibody-treated dogs’ serum.

Summary of potential adverse events with the initial cPD-L1 antibody administration to laboratory dogs

The list of genes that were altered in the activated canine PBMCs from the nCounter canine immune-oncology panel analysis

OBJECTIVE To investigate the role of omega-3 polyunsaturated fatty acids (Ω-3)–derived proresolving lipid mediators (PRLM) in the resolution of mild airway inflammation in horses. ANIMALS 20 horses with mild airway inflammation. PROCEDURES Horses previously eating hay were fed hay pellets (low Ω-3 content; n = 10) or haylage (high Ω-3 content; 9) for 6 weeks. Dust exposure was measured in the breathing zone with a real-time particulate monitor. Bronchoalveolar lavage (BAL) was performed at baseline, week 3, and week 6. The effect of PRLM on neutrophil apoptosis and efferocytosis was examined in vitro. BAL fluid inflammatory cell proportions, apoptosis of circulating neutrophils, efferocytosis displayed by alveolar macrophages, and plasma lipid concentrations were compared between groups fed low and high amounts of Ω-3 by use of repeated measures of generalized linear models. RESULTS Dust exposure was significantly higher with hay feeding, compared to haylage and pellets, and equivalent between haylage and pellets. BAL fluid neutrophil proportions decreased significantly in horses fed haylage (baseline, 11.8 ± 2.4%; week 6, 2.5 ± 1.1%) but not pellets (baseline, 12.1 ± 2.3%; week 6, 8.5% ± 1.7%). At week 6, horses eating haylage had significantly lower BAL neutrophil proportions than those eating pellets, and a significantly lower concentration of stearic acid than at baseline. PRLM treatments did not affect neutrophil apoptosis or efferocytosis. CLINICAL RELEVANCE Despite similar reduction in dust exposure, horses fed haylage displayed greater resolution of airway inflammation than those fed pellets. This improvement was not associated with increased plasma Ω-3 concentrations. Feeding haylage improves airway inflammation beyond that due to reduced dust exposure, though the mechanism remains unclear.

Demonstration of the

Well defined detection and analysis of nanoparticle-sized samples such as extracellular vesicles or viruses may be important for potential disease diagnostics. However, using conventional flow-cytometry optical methods to evaluate such small particles is quite challenging. The reason is that the particle is smaller than the diffraction limit, making detection difficult. An alternative approach is fluorescence detection via conjugated fluorochromes attached to the nanoparticles; the challenge in this case is the limitation imposed upon detection of a very small number of emitted photons buried in high background photon counts. Emitted fluorescence is described by the well-known equation kf = σa I Q, which describes the emitted fluorescence rate (kf) (photons/s) as the multiplication of molecular absorption cross section(σa), excitation intensity (I), and quantum yield (Q). In addition, the excitation rate is equal to 1/t, which is the inverse of the lifetime of several ns representing the most typical conjugated fluorescent molecules used in flow cytometry. We recently developed a sub-ns photon sensor that is faster than most fluorescence lifetimes, since sub-ns speed is a critically important parameter for the separation of individual emitted photons. Based on our observation of fluorescence and background levels on typical commercial flow cytometers it is evident that a significant component of the background is induced by water-molecular vibrations. Therefore, understanding what constitutes all the components that contribute to the signals we measure in flow cytometry would help in defining what we currently call "background signals." We attempted to define a theoretical model to try to unravel these issues. This model was based on use of a reflective dry surface in the absence of water molecules. Our objective was to determine if it is possible to minimize background and enhance signal, and to provide valuable information on the contributing components of the signals collected. In order to test this model, we tested a single dried particle 50 nm in diameter on a reflective surface with minimum background. While this is clearly not a standard biological system, our results suggest that this quantum approach closely follows established photon base theory. Our goal was to define the parameters for practical nanoparticle-fluorescence analysis while enhancing our knowledge of the contribution of background properties.

This study explores the adoption of laser-induced breakdown spectroscopy (LIBS) for the analysis of lateral-flow immunoassays (LFIAs). Gold (Au) nanoparticles are standard biomolecular labels among LFIAs, typically detected via colorimetric means. A wide diversity of lanthanide-complexed polymers (LCPs) are also used as immunoassay labels but are inapt for LFIAs due to lab-bound detection instrumentation. This is the first study to show the capability of LIBS to transition LCPs into the realm of LFIAs, and one of the few to apply LIBS to biomolecular label detection in complete immunoassays. Initially, an in-house LIBS system was optimized to detect an Au standard through a process of line selection across acquisition delay times, followed by determining limit of detection (LOD). The optimized LIBS system was applied to Au-labeled Escherichia coli detection on a commercial LFIA; comparison with colorimetric detection yielded similar LODs (1.03E4 and 8.890E3 CFU/mL respectively). Optimization was repeated with lanthanide standards to determine if they were viable alternatives to Au labels. It was found that europium (Eu) and ytterbium (Yb) may be more favorable biomolecular labels than Au. To test whether Eu-complexed polymers conjugated to antibodies could be used as labels in LFIAs, the conjugates were successfully applied to E. coli detection in a modified commercial LFIA. The results suggest interesting opportunities for creating highly multiplexed LFIAs. Multiplexed, sensitive, portable, and rapid LIBS detection of biomolecules concentrated and labeled on LFIAs is highly relevant for applications like food safety, where in-field food contaminant detection is critical. Graphical abstract.

Oral health conditions (eg, plaque, calculus, gingivitis) cause morbidity and pain in companion animals. Thus, developing technologies that can ameliorate the accumulation of oral biofilm, a critical factor in the progression of these conditions, is vital. Quantitative light-induced fluorescence (QLF) is a method to quantify oral substrate accumulation, and therefore, it can assess biofilm attenuation of different products. New software has recently been developed that automates aspects of the procedure. However, few QLF studies in companion animals have been performed. QLF was used to collect digital images of oral substrate accumulation on the teeth of dogs and cats to demonstrate the ability of QLF to discriminate between foods known to differentially inhibit oral substrate accumulation. Images were taken as a function of time and diet. Software developed by the Cytometry Laboratory, Purdue University quantified biofilm coverage. Intra- and intergrader reproducibility was also assessed, as was a comparison of the results of the QLF software with those of an experienced grader using undisclosed coverage-only metrics similar to those used for the Logan and Boyce index. Quantification of oral substrate accumulation using QLF-derived images demonstrated the ability to distinguish between dental diets known to differentially inhibit oral biofilm accumulation. Little variance in intra- and intergrader reproducibility was observed, and the comparison between the experienced Logan and Boyce grader and the QLF software yielded a concordance correlation coefficient of 0.89 (95% CI = 0.84, 0.92). These results show that QLF is a useful tool that allows the semi-automated quantification of the accumulation of oral biofilm in companion animals.