Biologics Safety and Disposition, Preclinical Safety, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Werk Klybeck, Basel, Switzerland.
Cytokine. 2012 Dec;60(3):828-37. doi: 10.1016/j.cyto.2012.08.018. Epub 2012 Sep 15.
The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats. We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which has so far received less attention in the scientific world with respect to the evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. Cytokine secretion into plasma or cell culture supernatants after 24h incubation with the test mAbs (anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release) was detected by a multiplex assay based on electrochemiluminescent excitation. We provide proof that this whole blood assay is a suitable new method for hazard identification of safety-relevant cytokine release in the clinic based on its ability to detect the typical cytokine signatures found in humans for the tested mAbs and on a markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™ - a clear advantage over the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine responses to the individual mAbs, in the concentration-response relationships and the prominent cytokine signatures for individual mAbs in the two formats reflect diverging mechanisms of cytokine release and different levels of dependency on high density coating even for two anti-CD28 super-agonistic antibodies. These results clearly show that one generic approach to assessment of cytokine release using in vitro assays is not sufficient, but rather the choice of the method, i.e. applying the whole blood assay or the PBMC assay needs to be well considered depending on the target characteristics and the mechanistic features of the therapeutic mAbs being evaluated.
已有数种单克隆抗体（mAb）被应用于人类，它们在使用过程中均与血液中细胞因子的显著释放相关联，进而导致急性不良反应。在这一领域中，毒理学物种的预测价值有限，这促使人们进行了大量的研究，以便建立使用外周血单个核细胞或血液的人源体外检测方法，从而预测人类的细胞因子释放。为了以最佳方式理解这些检测方法的预测价值，以识别危害并进行风险评估，并突出各个检测方法格式的潜在局限性，需要对这些检测方法进行全面的特征描述。我们对全血细胞因子释放检测方法进行了特征描述，该方法在水性检测体系中仅用测试抗体进行最小稀释（95%v/v 血液），与其他方法相比，这种方法在科学界对其是否适合预测人类细胞因子释放的评价中受到的关注较少。该方法与另一种已由他人充分描述的固定化 mAb 呈递的人 PBMC 检测方法进行了比较。在与测试 mAb（抗 CD28 超激动剂 TGN1412 类似物（TGN1412L）、另一种抗 CD28 超激动 mAb（ANC28.1）、T 细胞耗竭 mAb（Orthoclone™）和 TGN1412 等摩尔匹配对照物（Tysabri™））孵育 24 小时后，通过基于电化学发光激发的多重检测方法检测细胞因子分泌到血浆或细胞培养上清液中。我们提供的证据表明，由于该全血检测方法能够检测到测试 mAb 与人相关的典型细胞因子特征，并且由于背景检测值和与等摩尔匹配对照物 Tysabri™的细胞因子释放明显较低，因此该检测方法是一种合适的新方法，可用于识别临床相关细胞因子释放的安全性危害。与 PBMC 检测方法相比，这是一个明显的优势。重要的是，在两个检测方法中，对单个 mAb 的相对细胞因子反应、浓度反应关系和单个 mAb 的主要细胞因子特征的定量和定性差异，反映了细胞因子释放的不同机制以及对高密度包被的依赖性程度不同，即使对于两种抗 CD28 超激动剂抗体也是如此。这些结果清楚地表明，使用体外检测方法评估细胞因子释放的一种通用方法是不够的，而是需要根据目标特征和正在评估的治疗性 mAb 的机制特征，仔细考虑选择方法，即应用全血检测方法或 PBMC 检测方法。
