About InVivoMAb anti-mouse/human phosphorylated PD-1 (CD279) The 407.6G12 monoclonal antibody reacts with phosphorylated mouse PD-1. Specifically, the antibody only reacts with PD-1 when tyrosine 248 (Y248) is phosphorylated. PD-1 is a 50-55 kDa cell surface receptor that belongs to the CD28 family of the Ig superfamily. PD-1 is transiently expressed on CD4 and CD8 thymocytes as well as activated T and B lymphocytes and myeloid cells. PD-1 expression declines after successful elimination of antigen. PD-1 signals via binding its two ligands, PD-L1 and PD-L2. Upon ligand binding, PD-1 signaling inhibits T-cell activation, leading to reduced proliferation, cytokine production, and T-cell death. Phosphorylation of Y248 of the ITSM motif of PD-1 is essential for the initiation of inhibitory signals. PD-1 blockade leads to reduced phosphor-PD-1. The 6G12 antibody can be used to assess the efficacy of PD-1 pathway blockade. InVivoMAb anti-mouse/human phosphorylated PD-1 (CD279) Specifications IsotypeMouse IgG2a, κ Recommended Isotype Control(s)InVivoMAb mouse IgG2a isotype control, unknown specificity Recommended Dilution BufferInVivoPure pH 7.0 Dilution Buffer ImmunogenHuman PD-1 p248Tyr peptide conjugated to KLH Reported ApplicationsWestern Blot Flow Cytometry FormulationPBS, pH 7.0 Contains no stabilizers or preservatives Endotoxin<2EU/mg (<0.002EU/μg) Determined by LAL gel clotting assay Aggregation<5% Determined by DLS Purity>95% Determined by SDS-PAGE Sterility0.2 μm filtration ProductionPurified from cell culture supernatant in an animal-free facility PurificationProtein A Molecular Weight150 kDa StorageThe antibody solution should be stored at the stock concentration at 4°C. Do not freeze. Application ReferencesInVivoMAb anti-mouse/human phosphorylated PD-1 (CD279) (CLONE: 407.6G12)Bu X, Juneja VR, Reynolds CG, Mahoney KM, Bu MT, McGuire KA, Maleri S, Hua P, Zhu B, Klein SR, Greenfield EA, Armand P, Ritz J, Sharpe AH, Freeman GJ (2021). "Monitoring PD-1 Phosphorylation to Evaluate PD-1 Signaling during Antitumor Immune Responses" Cancer Immunol Res 9(12):1465-1475. PubMedPD-1 expression marks activated T cells susceptible to PD-1-mediated inhibition but not whether a PD-1-mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho-PD-1). We showed PD-1+ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1+TIM-3+ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8+ TILs. Phospho-PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.
