Pivotal Tumor Immunology Targets - PD-L1
New Rabbit mAbs for B7-H3 and B7-H4
Cancer Immunology Research article featuring
PD-L1 clone E1L3N®
The immune system employs a series of checkpoints to protect normal, healthy tissue from an immune response. These consist of receptors on the surface of activated T cells and their corresponding ligands on the surface of antigen presenting cells. A key immune checkpoint is created when PD-1 (programmed cell death protein 1) engages its ligand PD-L1. As a result of this interaction, T cell activation is attenuated and an active immune response is prevented(1).
This mechanism is also used by tumors. PD-L1 is upregulated in several tumor types and contributes to the malignancy of these cancers by interacting with PD-1 and inhibiting T cell activation. In this way, the tumors avoid detection and destruction by the immune system(1-3). Accordingly, PD-1 and PD-L1 have garnered much attention for their roles in tumor immunology and as potential immune-based therapeutic targets(4, 5).
PD-L1 Antibody - High Sensitivity
Detects endogenous levels of PD-L1 protein expression in human tissue
Immunohistochemical (IHC) analysis of paraffin-embedded human lung carcinoma using #13684. IHC was performed using SignalStain® Antibody Diluent #8112 and SignalStain® Boost IHC Detection Reagent #8114.
Recognizes PD-L1 and does not cross-react with other B7 family members
Western blot analysis of lysates from:
- COS cells transfected with PD-L2
- COS cells mock transfected
- KARPAS-299 cells
- SUP-M2 cells
Demonstrates consistent, reliable results in immunohistochemistry, flow cytometry, immunofluorescence, western blotting and immunoprecipitation.
Confocal IF analysis of paraffin-embedded human placenta using PD-L1 (E1L3N®) XP® Rabbit mAb #13684 (green) and Pan-Keratin (C11) Mouse mAb #4545 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of untreated SUP-M2 cells using PDL1 (E1L3N®) XP® Rabbit mAb #13864 (blue) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.
KARPAS-299 and PC-3 Cell Staining
|CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 1:200||Company 1 Rabbit mAb 1:8000||Company 2 Mouse mAb 1:50|
|Strong, membrane associated staining||Weaker membrane staining than #13684||No membrane staining|
|No staining||Diffuse, Non-specific staining||Diffuse, Non-specific staining|
IHC analysis demonstrates that CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 at 1:200 gives a strong signal in KARPAS-299, which are known to be PD-L1 high expressing cells, and no staining in PC-3 cells. In contrast, Company 1 Rabbit mAb and Company 2 Mouse mAb stain both KARPAS-299 and PC-3 cells meaning that they are not specific for PD-L1.
Lung and Breast Carcinoma
|CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 1:200||Company 1 Rabbit mAb 1:8000|
|Predominantly membrane staining in lung carcinoma and no staining in stromal cells.||Staining in cytoplasm and membrane in both lung carcinoma and stromal cells.|
|No staining in PD-L1 negative breast carcinoma.||Non-specific nuclear and cytoplasmic staining in the PD-L1 negative breast carcinoma.|
In IHC analysis of human lung and breast carcinoma CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 demonstrates appropriate staining in lung carcinoma with no staining in surrounding stromal cells, and no staining in breast carcinoma. Company 1 Rabbit mAb, however, demonstrates staining in both lung carcinoma and surrounding stromal cells, and inappropriate staining in breast carcinoma. This antibody is detecting a protein that is not PD-L1.
Western blot analysis using CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 results in detection of a band at the appropriate molecular weight for PD-L1 in KARPAS-299 and SUP-M2 cells, which are known to have high PD-L1 expression, and no protein in A549 and PC-3, cells demonstrating that #13684 is specific for PD-L1. At the dilution recommended by the supplier, Company 1 Rabbit mAb produces multiple signals at various molecular weights in A549, PC-3, KARPAS-299, and SUP-M2 cells, demonstrating that this antibody is not specific for PD-L1.
- Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12(4), 252–64.
- Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, Roche PC, Lu J, Zhu G, Tamada K, Lennon VA, Celis E, Chen L (2002) Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat. Med. 8(8), 793–800.
- Thompson RH, Kuntz SM, Leibovich BC, Dong H, Lohse CM, Webster WS, Sengupta S, Frank I, Parker AS, Zincke H, Blute ML, Sebo TJ, Cheville JC, Kwon ED (2006) Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up. Cancer Res. 66(7), 3381–5.
- Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, Drake CG, Camacho LH, Kauh J, Odunsi K, Pitot HC, Hamid O, Bhatia S, Martins R, Eaton K, Chen S, Salay TM, Alaparthy S, Grosso JF, Korman AJ, Parker SM, Agrawal S, Goldberg SM, Pardoll DM, Gupta A, Wigginton JM (2012) Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366(26), 2455–65.
- Brahmer JR, Pardoll DM (2013) Immune checkpoint inhibitors: making immunotherapy a reality for the treatment of lung cancer. Cancer Immunol Res 1(2), 85–91.