FIGURE 5 from Development of an Anti-canine PD-L1 Antibody and Caninized PD-L1 Mouse Model as Translational Research Tools for the Study of Immunotherapy in Humans

Evaluation of the caninized cPD-L1 chimeric antibody. A, 12C antibody binding on the BT549^cPD-L1 cells. B, Flow cytometric analysis of the 12C chimeric antibody on the BT549^cPD-L1 cells. cIgG serves as a negative control. C, 12C chimeric antibody binding to cPD-L1 and cPD-L2. D, Binding affinity (K_D) 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 (K9TCC^nRFP) 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 K9TCC^nRFP 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.