Recombinant hpd 1 mfc igg2a

Example 4

The ability of human anti-PD-L2 antibodies to inhibit the binding of human PD-1 fused with mouse-Fc protein (hPD-1-mFc) to CHO-K1 cells stably expressing human PD-L2 was evaluated by FACS. High antigen concentration (FACS): Individual anti-PD-L2 antibodies with serial dilution (0.25 ng/ml-4 μg/ml) were incubated with 1.5×10⁵ CHO-K1 cells expressing human PD-L2 for 30 min at 4° C. Unbound antibodies were removed and cells were washed two times with FACSwash buffer. Cells were resuspended in 50 ul FACSwash buffer containing 0.5 μg/ml recombinant hPD-1-mFc (IgG2a, BPS Bioscience) and incubated for 30 min at 4° C. Following two washes with FACSwash, cells were resuspended in 100 μl FACSwash containing PE-conjugated rat-anti-mouse IgG2a (Biolegend) at 1:200 dilution and incubated for 20 min at 4° C. in the dark. After three washes with FACSwash, bound hPD-1-mFc was detected and quantified by FACS analysis. Some wells were incubated with unspecific human or rat IgG with or without anti-mouse IgG to set minimal (0%) and maximal (100%) level of PD-L2/PD-1 binding. The percentage of maximal ligand binding in relation to the antibody concentration and IC50 values of each antibody were analyzed and visualized using non-linear curve fitting (GraphPad Prism Software) shown in FIG. 3.

Example 11

Comparison of the potency of fully human anti-PD-L2 antibodies with reference analogue anti-PD-L2 antibodies to inhibit the binding of human PD-1 fused with mouse-Fc protein (hPD-1-mFc) to CHO-K1 cells stably expressing human PD-L2 was evaluated by FACS. High antigen concentration (FACS): Individual anti-PD-L2 antibodies with serial dilution (0.25 ng/ml-4 μg/ml) were incubated with 1.5×10⁵ CHO-K1 cells expressing human PD-L2 for 30 min at 4° C. Unbound antibodies were removed and cells were washed two times with FACSwash buffer. Cells were resuspended in 50 ul FACSwash buffer containing 0.5 μg/ml recombinant hPD-1-mFc (IgG2a, BPS Bioscience) and incubated for 30 min at 4° C. Following two washes with FACSwash, cells were resuspended in 100 μl FACSwash containing APC-conjugated rat-anti-mouse IgG2a (Biolegend) at 1:200 dilution and incubated for 20 min at 4° C. in the dark. After three washes with FACSwash, bound hPD-1-mFc was detected and quantified by FACS analysis. Some wells were incubated with unspecific human IgG1 with or without anti-mouse IgG to set minimal (0%) and maximal (100%) level of PD-L2/PD-1 binding. IC50 values of each antibody were analyzed and visualized using non-linear curve fitting (GraphPad Prism Software). Table and bar graph including standard error are shown in FIG. 20.