Erinn Rankin and were authenticated by LabCorp (Burlington, NC
Erinn Rankin and were authenticated by LabCorp (Burlington, NC.) in March, 2021. alternative to PD-1 or PD-L1 therapeutic antibodies for achieving superior therapeutic efficacy in cancers expressing both PD-L2 and PD-L1. and ovarian cancer models, and is superior to antibodies targeting PD-L1 and PD-L2. Taken together, these studies indicate that PD-L2 plays a critical role in promoting an immune-suppressive microenvironment that can be overcome with the use of a high-affinity sPD-1 mutant. Materials and Methods Study design This study was designed to characterize structural, biochemical, functional and therapeutic efficacy of sPD-1 with high binding affinity to both PD-L1 and PD-L2 both experimentally and therapeutically. All tumor microarray specimens were commercially purchased from US Biomax (Derwood, MD). A board-certified veterinarian pathologist performed the Immunohistochemical scoring of the stained tumor microarray specimens. studies were conducted under the approval of AAAPLAC at Stanford University and ChemPartner, Shanghai. Sample sizes for animal studies were determined based on power calculations done on similar studies in previous studies. All animals were randomly assigned to treatment groups. Samples were not excluded from studies except for animals that required early termination due to illness that is unrelated to the study. Endpoints of experiments were defined in advance for each experiment. Tumor growth curves were presented for studies where tumor growth was measurable and Kaplan-Meier curves were used for orthotopic ovarian cancer models. Appropriate statistical analysis was used for each experimental study. Cell lines MC38, MC38 CRISPR Cells, ID8, ID8 CRISPR Cells, B16/OVA, MC38-hPD-L1, MC38-hPD-L2, Hep3B-hPD-L1, Hep3B-hPD-L2 and UPK10 cells were maintained in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in a humidified 37 C, 5% CO2 incubator. Cells were trypsinized and passaged at 80% confluency. VTX-2337 MC38 and Hep3B derived Cell lines were commercially obtained and authenticated through ChemPartner, Shanghai before the commencement of the studies. Early passages of ID8 and UPK10 cell lines were obtained from Dr. Erinn Rankin and were authenticated by LabCorp (Burlington, NC.) in March, 2021. PD-L1 (Cat no. sc-425636) and PD-L2 (Cat no. sc-425483) CRISPR KO construct were purchased through Santa Cruz Biotechnology (Dallas, TX) and performed according to manufacturer protocol. In vivo tumor studies All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Stanford University and animal ethical committee at ChemPartner, Shanghai. PD-L1 knockout mice on C57BL/6 background were kindly gifted by (Dr. Dean W. VTX-2337 Felsher at Stanford University). Female mice age 6C8 weeks were used for ID8 ovarian tumor studies. Mice were housed in pathogen-free animal facility, kept under constant temperature and humidity and controlled 12 h light-dark cycles. For ID8 and UPK10 ovarian studies including ID8 PD-L1 CRISPR KO, PD-L2 CRISPR KO and PD-L1/L2 CRISPR KO cells, 5106 or 20106cells were injected intraperitoneal or subcutaneously, animals terminated upon the Sirt6 development of peritoneal ascites for survival analysis or subcutaneous tumor reaches ethical termination point. For all ID8 tumor growth studies, ID8 cells were suspended in 50% Matrigel (#356230, Cornng, NY) and injected subcutaneously. Statistical analysis The Pearson correlation was used for all correlation analysis of tumor specimens. IHC H score values were determined by a board-certified veterinarian pathologist. All tumor volume, survival, quantification of immunohistochemistry were conducted using GraphPad Prism software (GraphPad Software Inc, CA). ANOVA with Tukey-Kramer test was used for comparing multiple treatment groups with each other. 0.05 was considered significant. Repeated measure ANOVA was used for comparing multiple treatment groups measured over time. Statistical analysis of survival curves was conducted for the VTX-2337 ID8 survival studies. A log-rank (Mantel-Cox) test was performed to compare mean survival among groups; 0.05 was considered statistically significant. Additional material and methods are available in the supplemental data Results PD-L2 is abundantly expressed in human ovarian cancers, but not in ICB sensitive bladder cancers. Although PD-L1 and PD-1 expression has been the subject of extensive study, less is known about the role of PD-L2 expression in cancer types that respond poorly to PD-1 or PD-L1 therapies (22, 23). To elucidate the clinical relevance of PD-L1 and PD-L2 expression in ovarian cancer, historically known to have a sub-optimal clinical response to ICB, we analyzed the expression of.