Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.
Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.
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DOI：
10.1038/s41379-019-0372-z
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发表时间：
2020-04
期刊：
影响因子：
--
通讯作者：
Barker C
中科院分区：
文献类型：
--
作者：
Lawson NL;Dix CI;Scorer PW;Stubbs CJ;Wong E;Hutchinson L;McCall EJ;Schimpl M;DeVries E;Walker J;Williams GH;Hunt J;Barker C
患者肿瘤样本中的程序性细胞死亡配体-1（PD-L1）表达水平已被证明在各种癌症类型中具有临床实用性。几种独立开发的PD-L1免疫组织化学（IHC）预测检测试剂盒已上市。使用VENTANA PD-L1（SP263）检测试剂盒、VENTANA PD-L1（SP142）检测试剂盒、Dako PD-L1 IHC 22 C3 pharmDx检测试剂盒、Dako PD-L1 IHC 28-8 pharmDx检测试剂盒和实验室开发的E1 L3 N抗体检测试剂盒的已发表研究（Cell Signaling Technology）已经证明了测定之间的PD-L1染色水平不同，导致推测抗体结合表位是否可能是导致测定之间不一致的原因。因此，为了了解不同PD-L1预测性免疫组织化学测定的性能，我们旨在区分PD-L1蛋白中负责抗体结合的表位。使用几种方法评估抗体克隆SP263、SP142、22 C3、28-8和E1 L3 N与重组PD-L1结合的位点，包括构象肽阵列、表面等离子体共振和/或氢/氘交换质谱法。通过PD-L1的定点诱变，随后通过表达突变体构建体的细胞系的蛋白质印迹和免疫组织化学分析来确认推定的结合位点。我们的结果表明，克隆SP263和SP142与PD-L1最末端C端胞质结构域中的相同表位结合，与22 C3和28-8不同。使用突变的PD-L1构建体，还发现另一个克隆E1 L3 N与PD-L1的胞质结构域结合。E1 L3 N结合表位与SP263/SP142结合位点相当大地重叠，但不相同。克隆22 C3和28-8在PD-L1的细胞外结构域中具有相互不同的结合特征。尽管鉴定了抗体之间的表位结合差异，但证据表明仅SP142测定产生显著不一致的免疫组织化学染色，这可以通过改变测定方案来解决。因此，试验间不一致性更可能归因于肿瘤异质性、试验或平台变量，而不是抗体表位。
Programmed cell death ligand-1 (PD-L1) expression levels in patient tumor samples have proven clinical utility across various cancer types. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available. Published studies using the VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, Dako PD-L1 IHC 22C3 pharmDx assay, Dako PD-L1 IHC 28-8 pharmDx assay, and laboratory-developed tests utilizing the E1L3N antibody (Cell Signaling Technology), have demonstrated differing levels of PD-L1 staining between assays, resulting in conjecture as to whether antibody-binding epitopes could be responsible for discordance between assays. Therefore, to understand the performance of different PD-L1 predictive immunohistochemistry assays, we aimed to distinguish the epitopes within the PD-L1 protein responsible for antibody binding. The sites at which antibody clones SP263, SP142, 22C3, 28-8, and E1L3N bind to recombinant PD-L1 were assessed using several methods, including conformational peptide array, surface plasmon resonance, and/or hydrogen/deuterium exchange mass spectrometry. Putative binding sites were confirmed by site-directed mutagenesis of PD-L1, followed by western blotting and immunohistochemical analysis of cell lines expressing mutant constructs. Our results demonstrate that clones SP263 and SP142 bind to an identical epitope in the cytoplasmic domain at the extreme C-terminus of PD-L1, distinct from 22C3 and 28-8. Using mutated PD-L1 constructs, an additional clone, E1L3N, was also found to bind to the cytoplasmic domain of PD-L1. The E1L3N binding epitope overlaps considerably with the SP263/SP142 binding site but is not identical. Clones 22C3 and 28-8 have binding profiles in the extracellular domain of PD-L1, which differ from one another. Despite identifying epitope binding variance among antibodies, evidence indicates that only the SP142 assay generates significantly discordant immunohistochemical staining, which can be resolved by altering the assay protocol. Therefore, inter-assay discordances are more likely attributable to tumor heterogeneity, assay, or platform variables rather than antibody epitope.
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PD-1和PD-L1检查点信号传导抑制癌症免疫疗法：机制，组合和临床结果。
影响因子：
5.6
作者：
Alsaab HO;Sau S;Alzhrani R;Tatiparti K;Bhise K;Kashaw SK;Iyer AK
通讯作者：
Iyer AK
影响因子：
28.4
作者：
Rimm DL;Han G;Taube JM;Yi ES;Bridge JA;Flieder DB;Homer R;West WW;Wu H;Roden AC;Fujimoto J;Yu H;Anders R;Kowalewski A;Rivard C;Rehman J;Batenchuk C;Burns V;Hirsch FR;Wistuba II
通讯作者：
Wistuba II
DOI：
10.1097/pai.0000000000000531
发表时间：
2018-02-01
影响因子：
1.6
作者：
Parra, Edwin R.;Villalobos, Pamela;Rodriguez-Canales, Jaime
通讯作者：
Rodriguez-Canales, Jaime
影响因子：
20.4
作者：
Hirsch, Fred R.;McElhinny, Abigail;Kerr, Keith M.
通讯作者：
Kerr, Keith M.
影响因子：
28.4
作者：
Gaule P;Smithy JW;Toki M;Rehman J;Patell-Socha F;Cougot D;Collin P;Morrill P;Neumeister V;Rimm DL
通讯作者：
Rimm DL