Gene targeting strategy for B-CAG-hPD-L1 B16-F10. The exon 3 of mouse PD-L1 gene that encode the extracellular domain was replaced by human PD-L1 CDS region sequence in B-CAG-hPD-L1 B16-F10.
Protein expression analysis
The PD-L1 expression analysis in B-CAG-hPD-L1 B16-F10 cells by flow cytometry. Single cells were collected from wild-type B16-F10 and B-CAG-hPD-L1 B16-F10 cells, and analyzed by flow cytometry with species-specific anti-PD-L1 antibody. Mouse PD-L1 was detectable in wild-type B16-F10 cells. Human PD-L1 was detectable in B-CAG-hPD-L1 B16-F10 cells but not wild-type B16-F10 cells.
Tumor Growth Curve & Body Weight Changes
Subcutaneous xenograft tumor growth of B-CAG-hPD-L1 B16-F10 cells. B-CAG-hPD-L1 B16-F10 cells and wild-type B16-F10 cells (1×105) were subcutaneously implanted into the C57BL/6 mice (female, 5-8 week-old, n=5).Tumor size and mice body weight were measured twice a week. (A) Tumor average volume ± SEM, (B) Mice body weight (Mean± SEM). Volume was expressed in mm3 using the formula: V=0.5a X b2, where a and b were the long and short diameters of the tumor, respectively. As shown in panel A, B-CAG-hPD-L1 B16-F10 were able to establish tumor in vivo and can be used for efficacy study.
PD-1 Ab (Keytruda) combined with OX40 Ab efficacy evaluation
The B16-F10-hPD-L1 cell line was subcutaneously implanted into homozygous B-hPD-1 / hOX40 mice. Mice were grouped when tumor size reached approximately 150 ± 50 mm3 (n = 5). (A) Mean tumor volume ± SEM; (B) Mean body weight of mice ± SEM.
The result shows a moderate antitumor activity by the Keytruda or OX40 Ab compared with the vehicle control group. The data also indicated that Keytruda combined with OX40 Ab potentiated the antitumor activity of the Keytruda or OX40 Ab alone.