5 different kinds of cytokine release assays: weathering the storm | CRA Post II - Insights From Our Labs to Yours

在我们以前的帖子中，我们概述了在与患者免疫系统相互作用的单克隆抗体（MAB）时，突出了细胞因子释放综合征（CRS）的危险和临床前细胞因子释放测定（CRAs）的重要性。yaboapp体育官网在第二个帖子中，我们描述了使用的不同类型，以及这些测定如何适合您的药物开发计划。在我们的下一个博客文章中，将更详细地讨论替代类型的CRA，外周血单核细胞（PBMC）血液产物细胞（BOEC）共培养。

Some of the questions you should consider when choosing the most appropriate CRA include the following:

Does the compound have an Fc region that can bind to neonatal Fc gamma receptors on endothelial cells?
该复合是否与多个目标结合？
目标表达健康受试者？
Which cytokines should you measure, since not all cytokines are upregulated during a cytokine storm?

Whole blood assays

Whole blood assays are one of the most widely used CRAs. Their main advantage is that they closely replicate in vivo conditions. The assay format recreates immune cell subset frequencies, FcγR cellular distribution and expression levels at physiological levels, revealing links between the FcγR genotype and magnitude of cytokine release in response to mAb treatment.

The whole blood CRA is far more sensitive than the PBMC solid-phase CRA (see below) for alemtuzumab (anti-CD52 antibody) which stimulates FcγRI-mediated cytokine release. However, whole blood CRAs are not sensitive toward muromonab-CD3 andTGN1412 (anti-CD28 superagonist)cytokine release. Other studies havedetermined在PBMC上优选整个血液测定格式，用于通过结合损伤的受体的寡核苷酸检测细胞因子释放。

Human PBMCs

PBMCS由纯分离淋巴细胞（T细胞，B细胞，NK细胞）和单核细胞组成。它们从全血中提取，因此测定比全血CRA在生理学上不那么生理，但可能对揭示细胞因子信号更敏感。

Solid-phase CRA, which involves the co-incubation of PBMCs with mAbs that have been dry-coated onto a tissue culture plate,was shownto be predictive for the cytokine release potential of TGN1412. In the liquid phase, PBMC assays do not produce target crosslinking or FC binding. Alternative solid-phase assessments include wet-coating mAbs onto the tissue culture well plastic at a lower density to that presented via dry-coating. All the above are used to mimic physiological mAb presentation but are ultimately different to in vivo presentation with unfortunate drawbacks with regards to false positive rate and high assay background. Dry and wet coat presentations also apply to the whole blood assay.

High Density CRA

First described by Romer et al in 2011在高密度预培养PBMC细胞提高试验的敏感性检测therapeutic T-cell activation. High density PMBCs respond akin to lymph node-like cells compared to regular low density PBMC assay. This assay doesn’t initiate target crosslinking or FC binding. Although unlike regular PBMC assays, this assay system can be used to detect positive responses to TGN1412 in a soluble/liquid phase due to the cells being in a “primed” lymph node-like state.

Human Umbilical Vein Endothelial Cell (HUVEC) PBMC Co-culture

This assay produces target cross-linking and FC binding. Endothelial cells are routinely grown from umbilical veins, which are used as the interface with PBMCs for cytokine release testing. Because these assays are made of a heterologous mixture of cells,这可能是结果in a tissue mismatch – when cells from one donor are mixed with those of another – and that this might be responsible for some of the limitations of HUVEC/PBMC co-culture assays.

Blood Outgrowth Endothelial cell (BOEC) PBMC Co-culture

This autologous assay produces target cross-linking and FC binding. First described by Mitchelle, et al, in 2015, this assay format is similar in concept to the HUVEC assay with a minor difference. BOECs are endothelial cells outgrown from PBMC collected from the same donor. Due to this assay being completely autologous, there are no tissue mismatch issues thus improving sensitivity of the assay. This assay system to date is the most reflective of in vitro systems to replicate the in vivo vascular microenvironment. A major benefit of this assay system over other formats is that the biologic therapeutic is presented in a more physiologically relevant manner akin to being dosed. This assay is a patented assay system and currently Covance is only CRO to commercially offer it.

典型的研究设计

由于产生的细胞因子的绝对水平根据所用的CRA测定而变化，因此可能难以识别真正的阳性结果。因此，细胞因子释放研究应包括相关的阳性和阴性对照。对照单克隆抗体的选择取决于评估产物的作用机制，例如，Muromonab-CD3或TGN1412用于T细胞靶向产品;Alemtuzumab和/或Rituximab用于具有细胞毒性效应功能的产品或植物原始对照的Phytohemaggluttin。

The study should also use blood products obtained from multiple donors (advise a minimum of 10) to enable a natural physiological mixture of samples. If target is not present in healthy donor samples, it is suggested to conduct hybrid studies using healthy donors alongside patient-derived samples. Suggestions for compound dosing selection should be selected based on the target receptor occupancy and as a minimum a high, medium and low dose should be considered.