Measuring IL-6 and sIL-6R in serum from patients treated with tocilizumab and/or siltuximab following CAR T cell therapy

Abstract

T cells expressing a CD19-specific chimeric antigen receptor (CAR19) are demonstrating remarkable efficacy in hematologic malignancies. Treatment is often associated with life-threatening cytokine release syndrome (CRS) which can be effectively treated with cytokine blockade using the antibodies, Siltuximab or Tocilizumab respectively targeting IL-6 or the IL-6 receptor. As IL-6 blockade is moving into the clinic for the treatment of CRS as well as IL-6-driven rheumatologic and malignant diseases, clinicians are utilizing serum cytokine panels more frequently to assess the effects of IL-6 inhibitors. It is paramount to ascertain whether levels obtained are accurate, especially as certain drugs may, in theory, affect quantification. We report the comparative quantification of IL-6 and sIL-6R using Luminex-based immunoassay kits from two vendors. Our results indicate good agreement of the commercial immunoassays in measurement of IL-6 but disagreement in quantitation of sIL-6R. We found that both Siltuximab and Tocilizumab can interfere with the measurement of their respective ligands using reagents from one vendor but not the second. This has significant implications for the analysis of IL-6 and sIL-6R pharmacokinetics analysis in Siltuximab or Tocilizumab-treated patients. We found that high levels of IL-6 can falsely reduce the measured levels of sIL-6R and high levels of sIL-6R can reduce levels of IL-6 when measured with some commercial assays. These data demonstrate the importance of assessing the impact of cytokine-blocking agents on accuracy of clinical biomarker assays in other diseases, as drugs targeting TNF-alpha, IL1B, and IL5 are being used more frequently in a large number of diseases.

Keywords: CAR-T; IL-6; Luminex; Siltuximab; Tocilizumab; sIL6-R.

Conflict of interest statement

Disclosure of conflicts of interest

Carl June, Stephan Grupp, David Teachey, Simon Lacey, J. Joseph Melenhorst, Noelle Frey, Shannon Maude, Fang Chen, Edward Pequignot, and David Porter received research funding from Novartis. Shannon Maude performs consultancy for Novartis. Carl June and David Porter have patents and royalties, and Stephan Grupp has a patent in the field of cell and gene therapies that are managed in accordance with University of Pennsylvania policy and oversight.

Copyright © 2016 Elsevier B.V. All rights reserved.

Figures

Fig. 1

Potential interaction among IL-6, sIL-6R, sgp130, Siltuximab, Tocilizumab, and xMAP antibody sets to IL-6, IL-6R and sgp130.

Fig. 2

IL-6 (A) and sIL-6R measurements on two serum samples using Millipore kit (open bar) and LifeTech kit (gray bar). Error bars are %CV from replicates. * shows the statistical difference between the measured values obtained with the two assay kits.

Fig. 3

Siltuximab, sIL-6R and Tocilizumab effect on IL-6 standard curves generated using Millipore kit (A) and Life Technologies kit (B).

Fig. 4

Siltuximab, Tocilizumab and sIL-6R effect on endogenous IL-6 concentration measurement in serum samples generated using Millipore (A) and LifeTech (B) kits. * shows the statistical difference in comparison to the corresponding control. Open box/bar — serum 1; gray box/bar — serum 2.

Fig. 5

Tocilizumab, IL-6 and Siltuximab effect on sIL-6R standard curves generated using Millipore kit (A) and LifeTech kits (B).

Fig. 6

Tocilizumab, Siltuximab and IL-6 effect on endogenous sIL-6R concentration measurement in serum samples generated using Millipore (A) and LifeTech (B) kits. Open box/bar — serum 3; gray box/bar — serum 4.

Fig. 7

IL-6, sIL-6R and sgp130 concentrations and fold changes, ferritin, temperature time course for UPCC04409–23. IL-6 fold-change is shown 10 fold lower than actual. IL-6 concentration is in pg/ml, while sIL-6R and sgp130 are in ng/ml.