A universal reporter cell line for bioactivity evaluation of engineered cytokine products

Abstract

christian pellegrino & Dario neri * engineered cytokine products represent a growing class of therapeutic proteins which need to be tested for biological activity at various stages of pharmaceutical development. in most cases, dedicated biological assays are established for different products, in a process that can be time-consuming and cumbersome. Here we describe the development and implementation of a universal cell-based reporter system for various classes of immunomodulatory proteins. the novel system capitalizes on the fact that the signaling of various types of pro-inflammatory agents (e.g., cytokines, chemokines, Toll-like receptor agonists) may involve transcriptional activation by NF-κB. Using viral transduction, we generated stably-transformed cell lines of B or T lymphocyte origin and compared the new reporter cell lines with conventional bioassays. The experimental findings with various interleukins and with members of the TNF superfamily revealed that the newly-developed "universal" bioassay method yielded bioactivity data which were comparable to the ones obtained with dedicated conventional methods. The engineered cell lines with reporters for NF-κB were tested with several antibody-cytokine fusions and may be generally useful for the characterization of novel immunomodulatory products. the newly developed methodology also revealed a mechanism for cytokine potentiation, based on the antibody-mediated clustering of TNF superfamily members on tumor-associated extracellular matrix components.

Targeting an engineered cytokine with interleukin-2 and interleukin-15 activity to the neovasculature of solid tumors

“…We tested cytokine activity using a recently reported NF-κB reporter assay in transduced murine CTLL-2 cells [ 45 ] ( Figure 2A ) and a proliferation assay with murine CTLL-2 cells (Supplementary Figure 1). Both assays indicated that the fusion proteins had similar cytokine activities, which was comparable to the one of the recombinant cytokine.…”

“…In general, the remaining cancer cells were cultured under a 5% CO 2 atmosphere at 37°C. CTLL-2 and transduced reporter CTLL-2 cells [ 45 ] were cultured in RPMI (Gibco) containing 10% FBS (Gibco), 1× antibiotic-antimycotic (Gibco), 2 mM Ultraglutamine (Lonza), 25 mM HEPES (Gibco), 50 μM β-mercaptoethanol (Sigma) and 60 U/mL IL-2 (Proleukin, Roche Diagnostics). F9 teratocarcinoma cells were cultured in flasks coated with 0.1% gelatin (Type B from Bovine Skin, Sigma), and they were cultured in DMEM (Gibco) containing 10% FBS (Gibco) and 1× antibiotic-antimycotic (Gibco).…”

“…The NF-κB reporter cell line was used according to the previous report [ 45 ]. Briefly, the reporter cells (transduced reporter CTLL-2) were starved for IL2 6-9 hours prior to use.…”

An engineered 4-1BBL fusion protein with “activity-on-demand”

“…The development of the CTLL-2 reporter cell line is described elsewhere 66 . CTLL-2_NF-B reporter cells were starved by washing the cells twice with prewarmed HBSS (Gibco, #14175095) followed by growth in the absence of IL-2 for 6 -9 h in RPMI 1640 (Gibco, # 21875034) medium supplemented with 10% FBS (Gibco, #10270106), 1 X antibioticantimycoticum (Gibco, # 15240062), 2 mM Ultraglutamine (Lonza, # BE17-605E/U1), 25 mM HEPES (Gibco, # 15630080) and 50 M -mercaptoethanol (Sigma Aldrich) in order to reduce the background signal.…”

Engineering murine GITRL for antibody-mediated delivery to tumor-associated blood vessels

“…The development of the CTLL-2 reporter cell line was described previously 36 . Briefy, CTLL-2 reporter cells were washed with prewarmed HBSS (Gibco, #14175095) and grown for 6 -9 h in growth medium without IL-2 prior to use in order to reduce the background signal.…”