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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. |
