Background

Durable tumor regressions are observed in a subset of advanced-stage non-small cell lung cancer (NSCLC) patients receiving PD-1/−L1 targeted immune checkpoint inhibitors (or ‘immunotherapy’) alone or in combination with chemotherapy. However, the majority of advanced NSCLC patients receiving these agents do not experience long-term disease control. Existing methods to identify patients most likely to gain clinical benefit from PD-1/−L1 immunotherapy have limitations, creating a need for improved methods to guide treatment selection, particularly for those likely to benefit from single-agent immunotherapy. Here, we describe the development of a series of novel assays for tumor-associated autoantibodies as part of an exploratory study intended to determine if these biomarkers have potential prognostic value in this setting.

Method

A selection of recombinant tumor autoantigens previously characterized for their diagnostic utility were developed and preliminarily evaluated by this study. These include: Fumarate Dehydrogenase (FH), Hydroxysteroid 17-Beta Dehydrogenase 10 (HSD17B10), Inosine Monophosphate Dehydrogenase 2 (IMPDH2), New York Esophageal Squamous Cell Carcinoma-1 (NY ESO-1), Phosphoglycerate Mutase 1 (PGAM1), and Vimentin. Custom Luminex immunobead assays were developed for these targets to quantitatively assess autoantibody levels in individual patient sera. Assays were erected as indirect immunoassays on MagPlex® Microspheres using standard carbodiimide/NHS-based chemistries, utilizing a biotin-conjugated secondary (i.e. anti-human IgG) antibody and R-phycoerythrin-conjugated streptavidin reporter system. Standard curves were created for quantitative purposes using commercially-available anti-antigen antibodies and permitted analytical performance characteristics to be calculated. These assays were used to preliminarily evaluate a series of pretreatment serum samples from stage IV NSCLC patients receiving anti PD-1/−L1 therapy after failure of at least one prior line of therapy (n = 40) and their classification efficiency calculated based on 12 months overall survival (OS) threshold.

Results

Six assays were developed that each showed dynamic ranges of four orders of magnitude and provided more than 90% classification accuracy based on the observed clinical outcome data. Inter- and intra-assay precision was assessed within these standards and overall %CVs of ≤7% and ≤ 10%, respectively, were calculated. Generally, the baseline level of autoantibodies were significantly (p < 0.05) lower in the ≥12 months survival group relative to the <12 months survival groups. Serum titers of FH, HSD170B, NY-ESO-1, and vimentin were significantly correlated with ≥12 month survival (p-value 0.0038, 0.0061, 0.0073, and 0.022, respectively). IMPDH2 and PGAM1 were found to have marginal significance (p-value 0.08 and 0.076, respectively).

Conclusion

This study demonstrates an efficient and promising means for assessing circulating autoantibody titers that could be useful in selecting advanced NSCLC patients for PD-1/−L1 directed immunotherapy. Further exploration and validation of this paradigm is warranted to further refine current treatment selection methods for this therapeutic strategy.

中文翻译：

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接受 PD-1/-L1 定向免疫检查点抑制的晚期 NSCLC 患者循环肿瘤相关自身抗体与临床结果之间的关系。

背景

在接受单独或联合化疗的 PD-1/-L1 靶向免疫检查点抑制剂（或“免疫疗法”）的晚期非小细胞肺癌 (NSCLC) 患者的子集中观察到持久的肿瘤消退。然而，大多数接受这些药物治疗的晚期 NSCLC 患者并未获得长期的疾病控制。识别最有可能从 PD-1/-L1 免疫疗法中获得临床益处的患者的现有方法存在局限性，因此需要改进方法来指导治疗选择，特别是对于那些可能从单药免疫疗法中受益的患者。在这里，我们描述了一系列针对肿瘤相关自身抗体的新型检测的开发，作为旨在确定这些生物标志物在这种情况下是否具有潜在预后价值的探索性研究的一部分。

方法

本研究开发并初步评估了一系列重组肿瘤自身抗原，这些抗原先前表征为它们的诊断效用。这些包括：富马酸脱氢酶 (FH)、羟基类固醇 17-β 脱氢酶 10 (HSD17B10)、肌苷单磷酸脱氢酶 2 (IMPDH2)、纽约食管鳞状细胞癌-1 (NY ESO-1)、磷酸甘油酸 PG 变位酶 1 (波形蛋白。针对这些目标开发了定制的 Luminex 免疫珠检测，以定量评估个体患者血清中的自身抗体水平。使用基于标准碳二亚胺/NHS 的化学试剂，利用生物素偶联的二级（即抗人 IgG）抗体和 R-藻红蛋白偶联的链霉亲和素报告系统，在 MagPlex® 微球上建立了间接免疫测定法。使用市售抗抗原抗体和允许的分析性能特征来创建用于定量目的的标准曲线以进行计算。这些测定用于初步评估来自接受抗 PD-1/-L1 治疗的 IV 期 NSCLC 患者的一系列治疗前血清样本，该患者在至少一个先前的治疗线失败后接受抗 PD-1/-L1 治疗。n  = 40) 以及基于 12 个月总生存 (OS) 阈值计算的分类效率。

结果

开发了六种检测，每种检测显示四个数量级的动态范围，并根据观察到的临床结果数据提供超过 90% 的分类准确度。在这些标准中评估了批间和批内精密度，并计算了总 %CV 分别≤7% 和≤10%。一般来说， 相对于 <12 个月生存组，≥12 个月生存组的自身抗体基线水平显着（p < 0.05）较低。FH、HSD170B、NY-ESO-1 和波形蛋白的血清滴度与≥12 个月的生存率显着相关（p值分别为 0.0038、0.0061、0.0073和 0.022）。发现 IMPDH2 和 PGAM1 具有边际显着性（p 值分别为 0.08 和 0.076）。

结论

该研究展示了一种评估循环自身抗体滴度的有效且有前景的方法，可用于选择晚期 NSCLC 患者进行 PD-1/-L1 定向免疫治疗。有必要进一步探索和验证这种范式，以进一步完善该治疗策略的当前治疗选择方法。