In the present study, we compare the specificity and sensitivity of the antibodies clone 28-8 and clone E1L3N, to evaluate the performance of the validated Bristol-Myers Squibb (BMS) and Dako assay (PD-L1 IHC 28-8 pharmDx) and the Cell Signaling Technology (CST) assay
In the present study, we compare the specificity and sensitivity of the antibodies clone 28-8 and clone E1L3N, to evaluate the performance of the validated Bristol-Myers Squibb (BMS) and Dako assay (PD-L1 IHC 28-8 pharmDx) and the Cell Signaling Technology (CST) assay. clone E1L3N, was compared with 28-8 for specificity and sensitivity using an identical detection method followed by vendor-recommended detection methods. Results Using PD-L1 null clones of L2987 and ES-2 tumor cell lines, both antibodies were specific for detection of PD-L1 on the plasma membrane, although E1L3N also stained cytoplasm in ES-2 Rabbit Polyclonal to MMP-19 knockout cells. Using the identical method, E1L3N was slightly more sensitive than EO 1428 28-8 based on staining intensities. Using manufacturer-recommended detection methods and predefined rating criteria for plasma membrane staining of tumor and immune cells, 28-8 shown significantly improved detection compared with E1L3N. Conclusions Epitope retrieval and highly sensitive detection reagents are key determinants in IHC detection of PD-L1. Electronic supplementary material The online version of this EO 1428 article (doi:10.1007/s40291-016-0237-9) contains supplementary material, which is available to authorized users. Key Points Rabbit monoclonal anti-programmed death-ligand 1 (antiCPD-L1) antibodies clone 28-8 and E1L3N both shown PD-L1 target specificity (E1L3N, only in the plasma membrane).Level of sensitivity of the two antibodies was comparable when an identical immunohistochemical retrieval and detection method was used; however, detection significantly improved with 28-8 versus E1L3N using manufacturer-recommended methods specific for each antibody.Epitope retrieval and sensitive detective reagents are important for achieving optimal target specificity and level of sensitivity. Open in a separate window Intro Nivolumab, a fully human being immunoglobulin (Ig) G4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody, is definitely authorized in the USA for unresectable or metastatic melanoma only or in combination with ipilimumab, advanced renal cell carcinoma (RCC) after prior antiangiogenic therapy, metastatic nonCsmall-cell lung malignancy (NSCLC) after progression on or after platinum-based chemotherapy, and relapsed or progressive classical Hodgkin lymphoma following autologous hematopoietic stem cell transplantation and brentuximab vedotin [1]. It is authorized in the EU for unresectable or metastatic melanoma only or in combination with ipilimumab, advanced RCC after previous therapy, and locally advanced or metastatic NSCLC after previous chemotherapy [2]. PD-1 and its ligands are checkpoint regulators in immune cells [3C6]. Programmed death-ligand 1 (PD-L1), one of the two PD-1 ligands, can also be indicated on the surface of tumor cells like a potential mechanism to engage PD-1 on the surface of the effector immune cells and evade an antitumor immune response [7C10]. The manifestation of PD-L1 has been reported on tumor cells in NSCLC and melanoma, among additional tumor types [10C16]. Immunoassays utilizing different main antibodies, assay types, and scoring approaches to assess the prevalence of positive PD-L1 manifestation in NSCLC, melanoma, and RCC have been reported [7, 8, 10, 16C18], although few of these reports possess directly compared the EO 1428 effect of antibody specificity and detection level of sensitivity [18]. Studies have shown that antibodies developed against PD-L1 display variable ability to detect PD-L1 in the cell plasma membrane compartment compared with the cytoplasm compartment [19, 20]. Some antibodies may not actually become wholly specific for PD-L1 [18]. In the present study, we compare the specificity and level of sensitivity of the antibodies clone 28-8 and clone E1L3N, to evaluate the performance of the validated Bristol-Myers Squibb (BMS) and Dako assay (PD-L1 IHC 28-8 pharmDx) and the Cell Signaling Technology (CST) assay. The PD-L1 IHC 28-8 pharmDx assay EO 1428 is definitely authorized by the US Food and Drug Administration (FDA) like a complementary diagnostic for non-squamous NSCLC and melanoma in the USA and CE designated in the EU. Materials and Methods Generation of Antibodies 28-8 and E1L3N, Tumor Cell Lines and Tumor Samples The rabbit monoclonal antihuman PD-L1 antibody 28-8 was produced by Abcam (lot #3), and the rabbit monoclonal antihuman PD-L1 antibody E1L3N by CST. Commercial tissue samples as well as L2987 and Sera-2 parent cell lines and their respective knockout cell lines, L2-14 and T1-11, were utilized for specificity screening. In-frame translation quit codons in L2987 and Sera-2 cells were introduced via genetic editing to produce clones of L2987 and Sera-2 that were null of PD-L1. A.