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

https://doi.org/10.1016/j.jim.2016.03.005Get rights and content

Highlights

  • IL-6 blockade is a strategy for the treatment of cytokine release syndrome as well as IL-6-driven diseases.

  • Serum cytokine panels being used clinically to assess the effects of IL-6 inhibitors.

  • Siltuximab and Tocilizumab therapy can interfere with the measurement of serum IL-6 and sIL-6R.

  • High levels of IL-6 can falsely reduce the measured levels of sIL-6R.

  • High levels of sIL-6R can reduce apparent levels of IL-6.

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.

Introduction

CAR19 T cells have demonstrated great promise in clinical trials, with clinical response rates of over 90% in children with relapsed/refractory ALL(Maude, SL, et al., 2015, Maude, SL, et al., 2014a, June, CH, et al., 2014). Encouraging response rates have also been seen in CLL(Porter et al., 2015) and NHL(Kochenderfer et al., 2015). These response rates are associated with considerable in vivo CAR T cell proliferation and clearance of CD19 + cells. However, killing of tumor cells is accompanied by cytokine release syndrome (CRS) (Porter et al., 2015). CRS can be mild with flu-like symptoms including fever, nausea, chills or life-threatening and severe with shock and respiratory compromise, leading to multi-system organ failure (Winkler et al., 1999). Markedly elevated levels of interleukin-6 (IL-6) and other cytokines drive CRS. Timely monitoring of cytokine levels, especially IL-6 can be extremely helpful in understanding CRS (Maude, SL, et al., 2014b, Xu, XJ and Tang, YM, 2014, Lee, DW, et al., 2014). We have recently described an algorithm for early prediction of severe CRS by monitoring serum cytokines (Teachey et al. Cancer Discovery, In Press).

IL-6 is a pro- and anti-inflammatory cytokine involved in various biologic processes. IL-6 has been associated with inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, vasculitis, and malignant and non-malignant lymphoproliferative disorders, including multiple myeloma and Castleman's syndrome (LH, Calabrese and Rose-John, S, 2014, Rossi, JF, et al., 2015). IL-6 is a small secreted glycoprotein composed of 184 amino acids, which form four helixes with a molecular weight ranging from 21 to 30 kD depending on the cells of origin(Simpson et al., 1997). It is produced by a range of cells under different conditions, such as stromal cells, hematopoietic cells, epithelial cells, or muscle cells (Rossi, JF, et al., 2015, Thompson, DK, et al., 2012, Wolf, J, et al., 2014). IL-6 concentrations are typically in the low picogram per ml range, in healthy individuals (Robak et al., 1998). However, IL-6 levels can elevate to nanograms per ml in patients with inflammation or infection, including sepsis (LH, Calabrese and Rose-John, S, 2014, Thompson, DK, et al., 2012, Robak, T, et al., 1998, Oda, S, et al., 2005).

Siltuximab, (CNTO 328, Sylvant) is a human-murine chimeric monoclonal antibody (MAb) against IL-6. It is a potent inhibitor of IL-6 with an estimated Kd of 1 pM (Zaki et al., 2004), and in 2014 was FDA-approved for the treatment of patients with multicentric Castleman's disease. Tocilizumab (Atlizumab, Actemra) is a humanized monoclonal antibody against IL-6R, which binds to both soluble and membrane-bound IL-6Rs with a Kd of ~ 2.54 nM (Mihara et al., 2005). It blocks IL-6 induced signal transduction pathways through competitive inhibition of IL-6 binding to its receptors (Nishina et al., 2013), and has been approved in Japan for treatment of Castleman's disease. Both Tocilizumab and Siltuximab have been used to treat CRS following CART19 therapy (Maude, SL, et al., 2014b, Grupp, SA, et al., 2013, Calabrese, LH and Rose-John, S, 2014).

There are several commercially available sandwich Enzyme-Linked Immuno-Sorbant Assay (ELISA)-based immunoassays for IL-6 and sIL-6R. Bead-based immunoassays, especially Luminex bead-based assays, due to their multiplexing capability, have replaced many of the traditional immunoassays (Leng et al., 2008). However, the analyte-specific antibodies and the recombinant protein used for the standard curve in each assay largely determine the specificity, reproducibility and sensitivity in either traditional microplate based or current bead based immunoassays (Butler, JE, et al., 1986, Vignali, DA, 2000). As a consequence, measurements of cytokine and receptor likely vary between vendors or even assay lots (Thompson, DK, et al., 2012, Ellington, AA, et al., 2010). We hypothesized that measurement of IL-6 or sIL-6R in serum could be altered by the presence of combinations of IL-6, sIL-6R, Tocilizumab, and/or Siltuximab (Fig. 1). IL-6 binds to the membrane-bound or soluble IL-6R with an affinity of around 1 nM (LH & Rose-John, 2014), thus IL-6 or sIL-6R can compete for binding with therapeutic or detection antibodies depending on the epitopes recognized by the antibodies.

In this study, we describe the results of a comparison of Luminex kits for human IL-6 and sIL-6R from EMD Millipore and Life Technologies to evaluate the effect of the presence of Siltuximab, sIL-6R, IL-6, and Tocilizumab on assay performance.

Section snippets

Materials

IL-6 (PHC0064), sIL-6R (10398-H08H-25) recombinant proteins, Luminex singleplex kits for IL-6 (LHC0061) and sIL-6R (LHR0061) were purchased from Life Technologies (Grand Island, NY). Another set of Luminex singleplex kits for IL-6 (HCYTOMAG-60K-01) and sIL-6R (HSCRMAG-32K-01) were purchased from EMD Millipore (Darmstadt, Germany).

Siltuximab (Janssen) and Tocilizumab (Genentech) were obtained from Children's Hospital of Philadelphia Pharmacy. Siltuximab was added to final concentrations of 20 

Evaluation of data derived from IL-6 and sIL-6R Luminex immunoassays from different vendors

IL-6 standard curves from Millipore and LifeTech both started from 5000 pg/ml with seven 1:3 serial dilution points (see Materials and method). Both curves had almost identical measured concentrations to the expected concentrations, which indicated close to 100% recovery at all seven concentrations with minimal %CV in duplicates (Supplemental Fig. 1A). sIL-6R standard curves from Millipore and LifeTech both started from 25,000 pg/ml with seven 1:3 serial dilution points (see Materials and method

Discussion

IL-6 exerts its biological functions via two major pathways: classic signaling and trans-signaling pathways. In the classic signaling pathway, IL-6 binds to the IL-6 receptor (IL-6R) on hepatocytes and some leukocytes. The IL-6_IL-6R complex further recruits the ubiquitously expressed membrane-bound or soluble gp130 (sgp130), triggering the dimerization of gp130 and intracellular signaling. In the trans-signaling pathway IL-6 interacts with soluble IL-6R (sIL-6R) to form the IL-6_sIL-6R

Authorship contributions

FC designed and conducted the experiments and prepared the manuscript. DTT reviewed the data and assisted in the writing. EP performed statistical analyses. NF, DP, SLM and SAG conducted the clinical trials of CTL019 and edited the manuscript. CHJ was the sponsor of the trials and advised on experimental design. JJM contributed to experimental plan design and edited the manuscript. SFL designed the experiments, and wrote and edited the manuscript.

Financial support was provided by a grant from

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.

Acknowledgments

We thank Natalka Kengle for the assistance with the Luminex cytokine assays, and Jeffrey Finklestein, Farzana Nazimuddin, Chelsie Bartoszek, Tatiana Mikheeva, Marina Bogush, and Patrick Landis for the sample processing. This study was supported by research funding from Novartis.

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