编程死亡配体1 (PD-L1) 抗体22C3是经批准的伴侣诊断免疫组化 (IHC) 检测，用于多种癌症的 Pemdrolizumab 和 Cemiplimab 治疗。22C3 和 28-8 抗体针对 PD-L1 的细胞外域 (ECD)，而 ECD 已知包含 N-糖基化位点。我们假设，当抗原的糖基化部分降解时，可能会影响其抗原性，并因此随着时间的推移而改变测定的评分。在这里，我们通过将细胞外域 (ECD) 抗原与细胞内域 (ICD) 抗原的抗体进行比较，测试样本的时间变化，并评估时间和去糖化对 PD-L1 信号的影响。我们选择了 2018 年的 10 个非小细胞肺癌 (NSCLC) 全组织切片进行测试。每个病例的新鲜切割序列切片均按照标签在 DAKO Link48 上用 22C3 进行染色。同时，使用先前描述过的实验室自主开发的 E1L3N (一种 ICD 抗体) 在 Leica BondRX 上进行了测试。病理学家读取了 22C3 和 E1L3N 的肿瘤分数 (TPS)，并将其与先前的临床诊断进行比较。为了确定使用定量方法的效果，我们同样评估了包含 90 个 NSCLC 病例的 TMA 考察组。最后，我们测试了标本的去糖化前后，以确定表位糖基化的可能影响是否存在。我们发现，与 3 年前的原始诊断样本相比，存档的 7/6 种阳性样本中的阳性染色显著减少。在一个较旧的存档 TMA 研究中，当使用 22C3 进行染色时，信号强度与 E1L3N 相比存在定量显著差异。这种信号的丧失未在新鲜的细胞系 TMA 中注意到，这与时间相关的染色降解有关。最后，当用 22C3 和 E1L3N 染色时，新鲜的 TMA 定量评估显示，去糖化过程会明显降低信号，而这种效应在使用 E1L3N 染色时没有被发现。我们认为这些数据表明，22C3 表位糖基化部分不稳定，并且在评估用于诊断或研究的存档组织时，应注意这个问题。Copyright © 2023. Published by Elsevier Inc.

Programmed death-ligand 1 (PD-L1)antibody 22C3 is the approved companion diagnostic immunohistochemistry (IHC) test for treatment with pembrolizumab and cemiplimab in multiple cancer types. The 22C3 and 28-8 antibodies target the extracellular domain (ECD) of PD-L1 which is known to contain N-glycosylation sites. We hypothesize that antigenicity could be affected by degradation of the glycan part of the epitope and thus change the scoring of the assay over time. Here, we test samples over time and assess the effects of time and deglycosylation on PD-L1 signal by comparing an antibody with an extracellular domain (ECD) antigen to an antibody with an intracellular domain (ICD) antigen. Ten whole tissue sections of non-small cell lung cancer (NSCLC) from 2018 were selected for testing. Fresh cut serial sections for each case were stained on DAKO Link48 for 22C3 according to the label. In parallel, a previously described laboratory developed test using E1L3N (an ICD antibody) was performed on the Leica BondRX. Tumor proportion scores (TPS) for 22C3 and E1L3N were read by a pathologist and compared to the previous clinical diagnoses. To determine the effect using a quantitative approach, a TMA cohort with 90 NSCLC cases was similarly assessed. Finally, to determine if the possible effect of epitope glycosylation, antibodies were tested before and after enzymatic deglycosylation of specimens. We found that 6/7 archival positive samples showed significant reduction in positive staining with 22C3 compared to original diagnostic sample assessed 3 years earlier. In an older archival TMA cohort, a quantitative significant difference in signal intensity was noted when staining with 22C3 was compared to E1L3N. This loss of signal was not noted in the fresh cell line TMA consistent with a time dependent degradation of staining. Finally, quantitative assessment of fresh TMA showed significant loss of signal after a deglycosylation procedure when stained with 22C3 which was not seen when stained with E1L3N. We believe this data shows that the glycan part of the 22C3 epitope is not stable over time, and that this issue should be considered when assessing archival tissue for diagnostic or research purposes.Copyright © 2023. Published by Elsevier Inc.