Research paperMeasuring IL-6 and sIL-6R in serum from patients treated with tocilizumab and/or siltuximab following CAR T cell therapy
Graphical abstract
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.
References (28)
- et al.
The immunochemistry of sandwich ELISAs–I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates
Mol. Immunol.
(1986) - et al.
Current concepts in the diagnosis and management of cytokine release syndrome
Blood
(2014) - et al.
CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia
Blood
(2015) - et al.
Tocilizumab inhibits signal transduction mediated by both mIL-6R and sIL-6R, but not by the receptors of other members of IL-6 cytokine family
Int. Immunopharmacol.
(2005) - et al.
Sequential measurement of IL-6 blood levels in patients with systemic inflammatory response syndrome (SIRS)/sepsis
Cytokine
(2005) - et al.
The interleukin 6 pathway and atherosclerosis
Lancet
(2012) Multiplexed particle-based flow cytometric assays
J. Immunol. Methods
(2000)- et al.
Cytokine-release syndrome in patients with B-cell chronic lymphocytic leukemia and high lymphocyte counts after treatment with an anti-CD20 monoclonal antibody (rituximab, IDEC-C2B8)
Blood
(1999) - et al.
Interleukin-6 and its receptors: a highly regulated and dynamic system
Cytokine
(2014) - et al.
Cytokine release syndrome in cancer immunotherapy with chimeric antigen receptor engineered T cells
Cancer Lett
(2014)
IL-6 biology: implications for clinical targeting in rheumatic disease
Nat. Rev. Rheumatol.
Antibody-based protein multiplex platforms: technical and operational challenges
Clin Chem
Chimeric antigen receptor-modified T cells for acute lymphoid leukemia
N Engl J Med
Cited by (133)
Senescent cells and SASP in cancer microenvironment: New approaches in cancer therapy
2023, Advances in Protein Chemistry and Structural BiologyThe effect of tocilizumab on patient reported outcomes and inflammatory biomarkers in hematopoietic cell transplantation
2022, Brain, Behavior, and Immunity - HealthClinical Presentation, Risk Factors, and Outcomes of Immune Effector Cell-Associated Neurotoxicity Syndrome Following Chimeric Antigen Receptor T Cell Therapy: A Systematic Review
2022, Transplantation and Cellular TherapyCitation Excerpt :Among the studies reporting coadministration of tocilizumab and steroids for ICANS, these patients also had concomitant CRS. Based on prior studies, using tocilizumab to manage ICANS in the absence of concurrent CRS is not recommended owing to its poor blood-brain barrier penetration and concerns about worsening ICANS related to tocilizumab's ability to cause elevations in cerebrospinal fluid IL-6 levels [9,37-39]. Few studies included in our systematic review evaluated long-term neurologic outcomes following CAR T cell therapy.
Important Considerations in the Intensive Care Management of Acute Leukemias
2024, Journal of Intensive Care Medicine