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Development of an in vitro potency bioassay for therapeutic IL-13 antagonists: The A-549 cell bioassay
Journal of Immunological Methods 334 (2008) 134 – 141 www.elsevier.com/locate/jim
Research paper
Development of an in vitro potency bioassay for therapeutic IL-13 antagonists: The A-549 cell bioassay Renee Miller a , Ramkrishna Sadhukhan b , Chengbin Wu a,⁎ b
a Department of Biologics, Abbott Bioresearch Center, Worcester, MA 01605, United States Department of Molecular & Cell Biology, Abbott Bioresearch Center, Worcester, MA 01605, United States
Received 25 October 2007; received in revised form 13 February 2008; accepted 14 February 2008 Available online 7 March 2008
Abstract Interleukin 13 is a key cytokine that mediates airway hyper-responsiveness and mucus over-production, and several anti-IL-13 therapeutic antibodies are currently in clinical development for the treatment of asthma. Conventional cell-based bioassays for evaluating neutralization potencies of IL-13 antagonists are semi-quantitative or with a low sensitivity. Here, we report the development of a highly sensitive bioassay to assess the potency of IL-13 antagonists using human lung epithelial A-549 cells that produce thymus and activation-regulated chemokine (TARC) in response to IL-13 stimulation. The A-549 cells were responsive to both wild type and a variant form of recombinant human IL-13 in a concentration-dependent manner, with a 16 to 24 h exposure time found to be within the linear portion of the bioassay response range. The Effective Concentration at 50% of the maximal response (ED50) of IL-13 determined for the assay was 1–5 ng/mL. With this level of IL-13, an anti-IL-13 antibody B-B13 yielded an approximate median Inhibitory Concentration (IC50) value of 0.2 nM. Bioassay optimization was performed to achieve best assay condition and sensitivity. Additionally, IL-13 antagonist potency against natural human IL-13 was also investigated in the A-549 cell bioassay. © 2008 Elsevier B.V. All rights reserved. Keywords: IL-13 antagonist; IL-13 antibody; TARC ELISA (Enzyme-Linked Immunosorbent Assay); In vitro; A-549; Potency; Bioassay
1. Introduction Human IL-13 is a Th-2 cytokine that signals through its cell surface receptors via signal transducer and activator of transcription (STAT6) and insulin receptor substrate-1/2 (IRS-1/2) pathways (Wang et al., 1995; Schnyder et al., Abbreviations: IL-13, interleukin 13; TARC, thymus and activationregulated chemokine; ED50, 50%; effective concentration; IC50, median inhibitory concentration; ATCC, American Type Culture Collection; BSA, bovine serum albumin. ⁎ Corresponding author. E-mail address: [email protected] (C. Wu). 0022-1759/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.02.009
1996; Takeda et al., 1996). In recent years, IL-13 has been implicated in the pathogenesis of human asthma as elevated levels of IL-13 mRNA and protein have been detected in lungs of asthmatic patients (Huang et al., 1995), and a variant form of human IL-13, resulted from a genetic polymorphism, has been associated with asthma and atopy (Heinzmann et al., 2000; Hoerauf et al., 2002; Vercelli, 2002; Heinzmann et al., 2003; Chen et al., 2004; Vladich et al., 2005). IL-13 has been validated as a target for asthma in preclinical animal models, as anti-IL-13 antibodies reduced airway hyperresponsiveness (AHR) and mucus hyper-secretion (Grunig et al., 1998; Wills-Karp et al.,
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1998; Taube et al., 2002). Currently, several therapeutic anti-IL-13 monoclonal antibodies are in pre-clinical and clinical development for the treatment of asthma. Bioassays can provide useful information regarding the drug activity or specificity in the search for potent and effective antagonists to IL-13. Moreover, with the numerous reports of IL-13 involvement in a variety of disease states, a sensitive bioassay system for measuring the biological activity of this cytokine and its antagonists have widespread applications. Several bioassay methods for measuring the activity of IL-13 and its antagonists have been previously reported. Human erythroleukemic TF-1 cells proliferate in response to IL-13 with a low signal-tobackground ratio (~2-fold) and an ED50 N= 100 ng/mL (Zurawski et al., 1993). Other assays, such as IL-13-induced CD23 expression on human B cells (Punnonen et al., 1993) and IL-13-induced expression of 15-lipoxygenase in human blood monocytes (Roy et al., 2002) are not quantitative, therefore cannot be used for precise measurements of potency of high affinity anti-IL-13 therapeutic monoclonal antibodies. IL-13 was previously reported to induce TARC production by the human lung epithelial adenocarcinoma cell line, A-549, in the presence of recombinant human TNF-alpha (rhTNF-α)(Yu et al., 2002; Faffe et al., 2003). This observation formed the basis of the bioassay discussed here, which provides several advantages over some of the bioassays previously mentioned. In addition, we also demonstrate the application of this assay in evaluating antibody potency against natural IL-13 secreted from human cells. Since IL-13 and IL-4 can signal through a common receptor system IL-13Rα1/IL-4R, and they are also co-produced/co-exist in many biological systems, it has been difficult to generate natural IL-13 without IL-4 also being produced simultaneously. Here we show that biologically active IL-13, but not IL-4, is produced from human PBMC under specific stimulation conditions, which could be applied to evaluating neutralization activity of therapeutic IL-13 antagonists. 2. Materials and methods Recombinant human IL-2, human IL-13 (wild type), mouse IL-13, rat IL-13, rhesus IL-13, anti-IL-13Rα1 polyclonal antibody (AF152), anti-13Rα2 polyclonal antibody (AF146), IL-13Rα1/Fc, IL-13Rα2/Fc fusion proteins (SF21 and NS0-derived), and human cytokine (IL-13, IL-4, and TARC) ELISA kits were purchased from R&D Systems. Anti-IL-13 monoclonal antibody clone B-B13 was purchased from Diaclone. Human variant IL-13 was purchased from PeproTech. Sheep and cynomolgus monkey IL-13 proteins were generated from
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transiently transfected COS-7 cells. A-549 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and were cultured in complete F12 growth medium (Gibco), containing 10% fetal bovine serum and supplemented with the 1% L-glutamine, 1% sodium bicarbonate, 50 units/mL penicillin, and 50 μg/mL Streptomycin. Mononuclear cell enriched leukocyte packs were obtained from Biological Specialty Corporation (Colmar, PA). 2.1. Optimizing the concentration of rhTNF-α for the A-549 bioassay A-549 cells were seeded 1 day prior to the experiment at 4 × 105 and 1 × 105 cells per well densities in a 96-well culture plate. On the day of the experiment, medium was replaced with100 μl fresh medium containing rhTNF-α (6.25 to 3.200 ng /mL). Additionally, rhIL-13 was also added to cells at a final concentration of 5 ng/mL. All well volumes were adjusted to 200 μL. After a 16–20 h overnight incubation, the supernatants were collected and TARC levels determined by ELISA. The optimum rhTNF-α concentration was determined by calculating the average TARC values, which were processed by GraphPad Prism. 2.2. Determining the A-549 cell density A-549 cells were plated 1 day prior to the experiment at four different seeding densities: 4 × 105, 2 × 105, 1 × 105 and 0.5 × 105 cells per well in 96-well culture plates. On the day of the experiment, medium was replaced with100 μl fresh medium containing rhTNF-α (the final concentration of rhTNF-α was 200 ng/mL). Additionally, rhIL-13 was also added to cells at a final concentration of 5 ng/mL. All well volumes were adjusted to 200 μL. After a 16–20 h overnight incubation, the supernatants were collected and TARC levels determined by ELISA. The optimum rhTNF-α concentration was determined by calculating the average TARC values, which were processed by GraphPad Prism. 2.3. Biological activity of wild type and variant rhIL-13, as well as IL-13 of other species in the A-549 bioassay A-549 cells were plated 1 day prior to the experiment at 2 × 105 cells per well. On the day of the experiment, medium was replaced with100 μl fresh medium containing rhTNF-α (the final concentration of rhTNF-α was 200 ng/mL). Subsequently, recombinant human (wt and variant), mouse, rat, sheep, rhesus and cynomolgus monkey IL-13 in fresh complete medium was added to
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media, and plated into 4 × 150 mm plate (40 ml/plate). In two plates, 400 ng recombinant human IL-4 (rhIL-4) and 200 µg anti human IL-12 (MAB219, R&D Systems) were added to induce a Th2 condition and marked as “skewed”. The other two plates were without rhIL-4 and anti-IL-12 and labeled as “unskewed”. The cells were cultured for 3 days. On day 4, cells from each individual plate were collected and resuspended in 40 ml complete media containing 10 ng/ml recombinant human IL-2, followed by incubating for additional 7 days to allow the cells to expand. On day 8, cells were counted and seeded into 24-well plate at 1.6 × 106 cells/well in 1 ml of complete media, then activated to produce cytokines (IL-13) by treatment with or without PMA and ionomycin. After 48 h, the IL-13-containing supernatants were harvested and cytokine levels determined by ELISA. The conditional media containing natural IL-13 were able to induce TARC in A-549 bioassay, and were further analyzed for antibody inhibition.
Fig. 1. Development and optimization of A-549 bioassay. A, Recombinant hTNF-α was titrated in the presence of rhIL-13 (5 ng/ mL) at 2 different cell densities, 1 ×105/well (○) and 4×105/well (■) (n=4, ⁎pb 0.01 between the 2 cell density groups). B, A-549 cells were further titrated at four different seeding densities: 4×105, 2×105, 1×105 and 0.5×105 cells/well in the presence of rhTNF-α (200 ng/mL) and rhIL13 (5 ng/mL). TARC production was measured by ELISA after a 16-h stimulation (n=4, ⁎pb 0.01 compared to the lowest cell density group). Data shown are representative of 3 independent experiments, and each data point was represented by quadruple wells.
each well, with the final concentration of IL-13 ranged from 0.015 to1000 ng/mL. After a 16–20 h overnight incubation, the supernatants were harvested and TARC levels determined by ELISA. EC50 values were calculated by GraphPad Prism software. 2.4. Generation of natural human IL-13 from PHA blast cells Human PHA Blasts were generated from lymphocytes (isolated from mononuclear cell enriched leukocyte packs) as previously described (Keen et al., 2006). PHA-blasted cells (1 × 108 cells/vial) were quickly thawed and mixed together, washed once with RPMI complete media, resuspended in 160 ml of complete
Fig. 2. IL-13 induces TARC production by A-549 cells in the presence of TNF-α. A, Recombinant wt (■) and variant (○) hIL-13 were titrated in the presence of rhTNF-α (200 ng/mL) at 2 × 105/well cell density. B, IL-13 of other species, including rat IL-13 (□), mouse IL-13 (▼), sheep IL-13 (▲), rhesus monkey IL-13 ( ), and cynomolgus monkey IL-13 ( ), can also induce TARC production, with potencies similar to human IL-13. TARC production was measured by ELISA after a 16-h stimulation. Data shown are representative of 3 independent experiments, and each data point was represented by quadruple wells (n = 4).
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2.5. Antibody Neutralization of recombinant and natural hIL-13 A-549 cells were plated 1 day prior to the experiment at 2 × 105 cells per well. On the day of the experiment, medium was replaced with100 μl fresh medium containing rhTNF-α (the final concentration of rhTNF-α was 200 ng/mL). The plates were placed at 37 °C, 5% CO2 until addition of mixture of human IL-13 (purified recombinant human IL-13 or nature IL-13 conditional medium) and IL-13 antagonists. Human IL-13 in complete F12 media (50 μL at 20 ng/mL) was mixed with IL-13 antagonists (50 μL from 1 pM to 5 μM), and the mixture was added to the rhTNF-α treated A-549 cells. All well volumes were equal to 200 μL. The final concentration of human IL-13 was 5 ng/mL. After a 16– 20 h incubation, the supernatants were harvested and TARC levels determined by ELISA. Neutralization potencies of IL-13 antagonists were calculated based on the average TARC values for each data point and the IC50 values determined by GraphPad Prism software.
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2.6. Statistical analysis ANOVA with post hoc Bonferroni correction was used to perform statistical analysis. Data are expressed as the mean ± SEM and a p b 0.05 was considered to indicate significant differences. 3. Results 3.1. Developing A-549 bioassay Several important assay parameters were optimized to establish an IL-13 bioassay with a high sensitivity. Recombinant hTNF-α was titrated (1600 ng/mL– 6.25 ng/mL) in the presence of a fixed amount of rhIL13 (5 ng/mL) and at different A-549 seeding cell densities. The final concentration of IL-13 at 5 ng/mL was determined from earlier work on IL-13 titration in the presence of several different hTNF-α concentrations (IL-13 ED50 ~ 1–5 ng/mL). Under these conditions, 200 ng/mL hTNF-α induced the maximum TARC
Fig. 3. Bioactive natural human IL-13 production from PHA-P blast cells. Human PBMC cells were PHA-P treated and incubated with or without PMA or ionomycin, under Th2-skewed (black bars) or unskewed (hatched bars) conditions, followed by expansion in the presence or absence of rhIL-2. Natural human IL-13 (A) and IL-4 (B) productions were measured by ELISA after a 48-h expansion. Data shown are pooled from 3 independent experiments using cells from 2 different donors, and each data point was represented by quadruple wells. There was no statistical difference between skewed and unskewed conditions for all groups except two (indicated by ⁎p b 0.01). The bioactivity of natural IL-13 was analyzed in the A-549 bioassay in comparison to E. coli-derived recombinant human wt IL-13 (both at 5 ng/mL) (C). Both induced similar amount of TARC without statistical difference.
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production by the A-549 cells, as additional rhTNF-α did not further increase the overall TARC signal (a representative experiment is shown in Fig. 1A). Subsequently, A-549 cell densities were titrated at 4 × 105, 2 × 105, 1 × 105 and 0.5 × 105 cells per well in the presence of 200 ng/mL rhTNF-α and 5 ng/mL hIL-13. As shown in Fig. 1B, cell density at 2 × 105 cells per well produced the maximum TARC levels, and there was no further increase in TARC production by increasing the cell number from 2 × 105 to 4 × 105 cells/well. Lastly, recombinant wt and variant IL-13 was titrated (1000 ng/mL–0.15 ng/mL) in the presence of a fixed amount of rhTNF-α at 200 ng/mL and cell density at 2 × 105 cells per well. TARC production increased in response to increasing concentrations of IL-13, with ED50 values in the range of 1-5 ng/mL for both wt and variant IL-13 (Fig. 2A). Consequently, hIL-13 concentration of 5 ng/mL was selected for subsequent neutralization assays. In addition, A-549 cells also responded to mouse, rat, sheep, cynomolgus, and rhesus IL-13, with ED50 values similar to that of human IL-13 (Fig. 2B). Culture supernatants from A-549 cells treated with medium alone or medium with 200 ng/mL rhTNF-α did not induce detectable levels of TARC. A-549 cells up to passage #55 (post acquisition from ATCC) have been used in this assay with consistent signal of response and reproducibility.
did not produce high levels of IL-13 (Fig. 3A), and subsequently did not induce detectable levels of TARC in the A-549 bioassay. 3.3. Determining neutralization potency of recombinant human IL-13 (rhIL-13) antagonists IL-13 antagonists were titrated (20 μg/mL–0.0003 μg/mL) in the presence of a fixed amount of rhTNF-α (200 ng/mL) and hIL-13 (5 ng/mL). Under these conditions, the concentration-dependent inhibition of TARC production by various IL-13 antagonists is shown in Fig. 4. Anti-IL-13 monoclonal antibody B-B13 and IL-13Rα2/Fc (SF21 cell-derived) exhibited high potencies in inhibiting IL-13-induced TARC production, with IC50 values of 0.2 and 0.7 nM, respectively. Interestingly, NS0-derived IL-13Rα2/Fc was less potent than that produced by SF21 cells. Both IL-13Rα1/Fc fusion protein and an anti-IL-13Rα1 polyclonal antibody showed low potencies in this assay (IC50 N 10 nM). In addition, an antiIL-13Rα2 polyclonal antibody did not show any inhibitive activity. Collectively, these data indicate that IL-13-induced TARC production by A-549 cells is mediated through IL-13Rα1/IL-4 receptor complex. This is consistent with the observation that IL-4 could also induce TARC production by A-549 cells concentration-dependently in the presence of TNF-α, with an ED50
3.2. Natural human IL-13 production from PHA blast cells Natural IL-13 was produced by using mitogen (PHA) activated human peripheral mononuclear cells. No significant difference in production of IL-13 was observed between Th2-skewed and unskewed conditions(Fig. 3A). PMA stimulation of IL-2-treated cells induced 10–15 ng/mL IL-13 production, and the presence of ionomycin did not further enhance IL-13 levels (Fig. 3A). In addition, the presence of ionomycin induced high levels of IL-4 under both Th2-skewed and unskewed conditions (Fig. 3B), indicating that activation of the PKC pathway was sufficient for IL-13 secretion, whereas activation of calcium signaling pathways was required for IL-4 production. There was no significant production of TARC under any of the conditions tested (data not shown). Therefore, in the absence of ionomycin, the lack of production of both IL-4 and TARC rendered the natural IL-13 produced suitable for testing in the A-549 bioassay. The natural IL-13 produced by this method at 5 ng/ml was able to induce TARC production by A-549 cells to similar levels as recombinant IL-13 (Fig. 3C). Culture supernatants from PHA Blasts treated with medium alone or PMA without IL-2
Fig. 4. Neutralization of rhIL-13-induced TARC production by IL-13 antagonists. Various IL-13 antagonists were titrated in the presence of recombinant human wt IL-13 (5 ng/mL) and rhTNF-α (200 ng/mL) at 2 × 105/well A-549 cell density. Antagonists included anti-IL-13 mAb B-B13 (■), rhIL-13Rα2/Fc (Sf21-derived) (△), rhIL-13Rα2/Fc (NS0-derived) ( ), anti-hIL-13Rα1 pAb (□), rhIL13Rα1/Fc (○), anti-hIL-13Rα2 pAb (▽), and a non-specific control mouse IgG (●). TARC production was measured by ELISA after a 16-h stimulation. Data shown are representative of 3 independent experiments, and each data point was represented by quadruple wells.
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Fig. 5. Neutralization of natural human IL-13-induced TARC production by IL-13 antagonists. IL-13 antagonists were titrated in the presence of natural human IL-13 (5 ng/mL) and rhTNF-α (200 ng/mL) at 2 × 105/well A-549 cell density. Antagonists included anti-IL-13 mAb B-B13 (■) and rhIL-13Rα2/Fc (□), and a non-specific control mouse IgG (●) is also included. TARC production was measured by ELISA after a 16-h stimulation. Data shown are representative of 3 independent experiments, and each data point was represented by quadruple wells.
of 0.1 nM (data not shown). In addition, anti-IL-13 mAb B-B13 and IL-13Rα2/Fc were able to neutralize naturally derived human IL-13 in a concentration-dependent manner (Fig. 5). The neutralization potencies (IC50 = 0.4 and 1.1 nM for B-B13 and IL-13Rα2, respectively) were similar to that observed against E-coli-derived recombinant human IL-13 for both antagonists. Furthermore, both antagonists were able to inhibit TARC production to 100%, indicating all TARC production was due to the activity of natural IL-13, and there was no IL-4 contamination. By RT-PCR and DNA sequencing, the IL-13 produced by this particular donor was also determined to be wt IL-13. 4. Discussion A robust bioassay is instrumental for the selection and development of a therapeutic monoclonal antibody. This report demonstrates a new bioassay with a desirable sensitivity that can support selection and pre-clinical development of a therapeutic IL-13 antagonist. Several methods for measuring IL-13 and its antagonist activity have been described previously (Punnonen et al., 1993; Zurawski et al., 1993; Roy et al., 2002). These methods possess some common limitations, such as the requirement for high IL-13 concentrations, low signal-to-background ratio, even with radioactive endpoints. Therefore, a novel bioassay was developed in our lab to assess in vitro potency of IL-13 and IL-13 antagonists, which exhibited some advantages over previously described methods. The A-549 cell line was shown previously to be an IL-13 responsive cell line, releasing TARC upon
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exposure to IL-13 in the presence of TNF-α (Yu et al., 2002; Faffe et al., 2003). Building on this observation, we established a reproducible bioassay method with a simple endpoint for measuring potency of IL-13 antagonists. The A-549 cell potency assay provides a valuable tool for assessing neutralizing activity of various IL-13 antagonists, without the use of [3H]-thymidine or special laboratory equipment and reagents. The A-549 bioassay is based on the direct measurement of IL-13 induced TARC from A-549 cell supernatants using a standard commercial ELISA, therefore it can be performed routinely in most laboratories. The endpoint for the A-549 assay is quantitative, and the A-549 cells have been found to respond to IL-13 consistently over many passages. A-549 cells also respond to IL-13 of different species with similar sensitivity (similar ED50), including human (wt and variant), cynomolgus and rhesus monkey, mouse, rat, and sheep IL-13. This feature of the bioassay is important to enable analyzing species cross-reactivity of any therapeutic IL-13 antagonists for toxicological studies. In contrast, the conventional B9 cell-based bioassay responds to human IL-13 approximately 100fold less sensitively than to mouse IL-13 (Lauder and McKenzie, 2001). Most therapeutic antibodies are generated against recombinant target proteins, either by in vivo hybridoma technology or in vitro phage display technology. As various types of differences exist between recombinant and naturally derived proteins (i.e. structure, glycosylation, activity, etc), it is critical to assess whether the therapeutic antibody can also recognize naturally derived target protein equally well. To this end, we established an in vitro system to produce natural human IL-13. However, since A-549 cells also respond to IL-4 and perhaps other mediators, it was therefore critical to induce IL-13 production without the induction of IL-4 secretion. This would significantly simplify the assay, as no further purification step to remove the IL-4 would be required. However, producing high levels of natural IL-13 without IL-4 secretion has proven difficult, as these two cytokines are often co-produced in many biological systems, i.e. a Th2-polarized T cell cultural condition. In this report, natural IL-13 was produced by pre-activated human PBMCs in the presence of PMA, ionomycin, or in combination, followed by IL-2 stimulation, in both Th2skewed and unskewed conditions. Interestingly, IL-4 production required the presence of ionomycin, followed by IL-2 stimulation, in both Th2-skewed and unskewed conditions. This indicated that activation of calcium signaling and subsequent activation of protein kinase C was required for IL-4 production but not for IL-13 production in this particular system. Therefore, we have
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developed an in vitro system for producing natural IL-13 without IL-4 production. In addition, the concentrations of natural IL-13 produced in this system was much higher than those produced by previously described Hodgkin lymphoma cell lines, such as HDLM-2 (Skinnider et al., 2001). Furthermore, we have determined that HDLM-2 cells spontaneously released high concentrations of TARC (N 20 ng/106 cells/ml/day), therefore natural IL-13 produced by HDLM-2 cells could not be readily used for A-549 bioassay prior to removal of existing TARC. In contrast, our current system does not product TARC under any of the conditions tested. Consequently, complete inhibition of TARC was achieved in natural IL-13 neutralization assay with both an anti-IL-13 mAb (B-B13) and IL-13Rα2/Fc fusion protein, indicating all TARC had been produced by the A-549 cells in response to natural IL-13. We demonstrate here that the IL-13 induces TARC expression in a lung epithelial adenocarcinoma cells in vitro. Alone, neither IL-13 nor TNF-α was effective. The combination of IL-13 and TNF-α synergistically stimulated TARC production. Synergy between IL-13 and TNF-α has previously been demonstrated for the induction of TARC from human peripheral blood mononuclear cells (Nomura et al., 2002), human keratinocyte and fibroblast (Yu et al., 2002), human bronchial epithelial cells (Sekiya et al., 2000), and human airway smooth muscle cells (Faffe et al., 2003). Similar synergistic effect was also observed for eotaxin release from airway epithelial and smooth muscle cells (Matsukura et al., 2001; Moore et al., 2002). Our study indicated that TARC production from the A-549 cells was the result of the induction of TARC expression, and not due to the proliferation of A-549 cells. Although IL-13 could induce minor proliferation for certain lung epithelial cells (Booth et al., 2001), we in fact did not detect any significant increase in A-549 cell numbers after incubating with IL-13 and TNF-α under the current assay condition (data not shown). It has been suggested that the synergy between TNF-α and IL-13 on eotaxin release could be transcriptionally mediated, as the STAT-6-binding element in the eotaxin promoter overlaps with a consensus binding site for nuclear factor (NF)-κB, and that simultaneous binding of both STAT-6 and NF-κB to this region of the eotaxin promoter results in enhanced promoter activity (Matsukura et al., 1999). As CCchemokine (including both TARC and eotaxin) genes are clustered within the same chromosomal region, we speculate that IL-13/ TNF-α synergy in TARC induction could be similar to eotaxin induction. As TARC has been implicated in asthma (Kawasaki et al., 2001; Berin, 2002; Sugawara et al., 2002), and has been suggested to be a
potential biomarker for anti-IL-13 therapeutics (Syed et al., 2007), this bioassay could be potentially used to measure biological response of IL-13 antagonists in preclinical and clinical settings. Acknowledgement We thank Megan Staples, Lori Dowding, Ruth Febo, and Romma Southwick of Abbott Bioresearch Center for their technical assistance. References Berin, M.C., 2002. The role of TARC in the pathogenesis of allergic asthma. Drug News Perspect. 15, 10. Booth, B.W., Adler, K.B., Bonner, J.C., Tournier, F., Martin, L.D., 2001. Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-alpha. Am. J. Respir. Cell Mol. Biol. 25, 739. Chen, W., Ericksen, M.B., Levin, L.S., Khurana Hershey, G.K., 2004. Functional effect of the R110Q IL13 genetic variant alone and in combination with IL4RA genetic variants. J. Allergy Clin. Immunol. 114, 553. Faffe, D.S., Whitehead, T., Moore, P.E., Baraldo, S., Flynt, L., Bourgeois, K., Panettieri, R.A., Shore, S.A., 2003. IL-13 and IL-4 promote TARC release in human airway smooth muscle cells: role of IL-4 receptor genotype. Am. J. Physiol., Lung Cell. Mol. Physiol. 285, L907. Grunig, G., Warnock, M., Wakil, A.E., Venkayya, R., Brombacher, F., Rennick, D.M., Sheppard, D., Mohrs, M., Donaldson, D.D., Locksley, R.M., Corry, D.B., 1998. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282, 2261. Heinzmann, A., Mao, X.Q., Akaiwa, M., Kreomer, R.T., Gao, P.S., Ohshima, K., Umeshita, R., Abe, Y., Braun, S., Yamashita, T., Roberts, M.H., Sugimoto, R., Arima, K., Arinobu, Y., Yu, B., Kruse, S., Enomoto, T., Dake, Y., Kawai, M., Shimazu, S., Sasaki, S., Adra, C.N., Kitaichi, M., Inoue, H., Yamauchi, K., Tomichi, N., Kurimoto, F., Hamasaki, N., Hopkin, J.M., Izuhara, K., Shirakawa, T., Deichmann, K.A., 2000. Genetic variants of IL-13 signalling and human asthma and atopy. Hum. Mol. Genet. 9, 549. Heinzmann, A., Jerkic, S.P., Ganter, K., Kurz, T., Blattmann, S., Schuchmann, L., Gerhold, K., Berner, R., Deichmann, K.A., 2003. Association study of the IL13 variant Arg110Gln in atopic diseases and juvenile idiopathic arthritis. J. Allergy Clin. Immunol. 112, 735. Hoerauf, A., Kruse, S., Brattig, N.W., Heinzmann, A., MuellerMyhsok, B., Deichmann, K.A., 2002. The variant Arg110Gln of human IL-13 is associated with an immunologically hyper-reactive form of onchocerciasis (sowda). Microbes Infect. 4, 37. Huang, S.K., Xiao, H.Q., Kleine-Tebbe, J., Paciotti, G., Marsh, D.G., Lichtenstein, L.M., Liu, M.C., 1995. IL-13 expression at the sites of allergen challenge in patients with asthma. J. Immunol. 155, 2688. Kawasaki, S., Takizawa, H., Yoneyama, H., Nakayama, T., Fujisawa, R., Izumizaki, M., Imai, T., Yoshie, O., Homma, I., Yamamoto, K., Matsushima, K., 2001. Intervention of thymus and activationregulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice. J. Immunol. 166, 2055. Keen, J.C., Cianferoni, A., Florio, G., Guo, J., Chen, R., Roman, J., Wills-Karp, M., Casolaro, V., Georas, S.N., 2006. Characterization
R. Miller et al. / Journal of Immunological Methods 334 (2008) 134–141 of a novel PMA-inducible pathway of interleukin-13 gene expression in T cells. Immunology 117, 29. Lauder, A., McKenzie, A.N.J., 2001. Measurement of interleukin-13. Current Protocols in Immunology, vol. 2. John Wiley & Sons, Hoboken, New Jersey, USA, p. 6.18.1. Matsukura, S., Stellato, C., Plitt, J.R., Bickel, C., Miura, K., Georas, S.N., Casolaro, V., Schleimer, R.P., 1999. Activation of eotaxin gene transcription by NF-kappa B and STAT6 in human airway epithelial cells. J. Immunol. 163, 6876. Matsukura, S., Stellato, C., Georas, S.N., Casolaro, V., Plitt, J.R., Miura, K., Kurosawa, S., Schindler, U., Schleimer, R.P., 2001. Interleukin-13 upregulates eotaxin expression in airway epithelial cells by a STAT6-dependent mechanism. Am. J. Respir. Cell Mol. Biol. 24, 755. Moore, P.E., Church, T.L., Chism, D.D., Panettieri Jr., R.A., Shore, S.A., 2002. IL-13 and IL-4 cause eotaxin release in human airway smooth muscle cells: a role for ERK. Am. J. Physiol., Lung. Cell. Mol. Physiol. 282, L847. Nomura, T., Terada, N., Kim, W.J., Nakano, K., Fukuda, Y., Wakita, A., Numata, T., Konno, A., 2002. Interleukin-13 induces thymus and activation-regulated chemokine (CCL17) in human peripheral blood mononuclear cells. Cytokine 20, 49. Punnonen, J., Aversa, G., Cocks, B.G., McKenzie, A.N., Menon, S., Zurawski, G., de Waal Malefyt, R., de Vries, J.E., 1993. Interleukin 13 induces interleukin 4-independent IgG4 and IgE synthesis and CD23 expression by human B cells. Proc. Natl. Acad. Sci. U. S. A. 90, 3730. Roy, B., Bhattacharjee, A., Xu, B., Ford, D., Maizel, A.L., Cathcart, M.K., 2002. IL-13 signal transduction in human monocytes: phosphorylation of receptor components, association with Jaks, and phosphorylation/activation of Stats. J. Leukoc. Biol. 72, 580. Schnyder, B., Lahm, H., Woerly, G., Odartchenko, N., Ryffel, B., Car, B.D., 1996. Growth inhibition signalled through the interleukin-4/ interleukin-13 receptor complex is associated with tyrosine phosphorylation of insulin receptor substrate-1. Biochem. J. 315 (Pt 3), 767. Sekiya, T., Miyamasu, M., Imanishi, M., Yamada, H., Nakajima, T., Yamaguchi, M., Fujisawa, T., Pawankar, R., Sano, Y., Ohta, K., Ishii, A., Morita, Y., Yamamoto, K., Matsushima, K., Yoshie, O., Hirai, K., 2000. Inducible expression of a Th2-type CC chemokine thymus- and activation-regulated chemokine by human bronchial epithelial cells. J. Immunol. 165, 2205.
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Skinnider, B.F., Elia, A.J., Gascoyne, R.D., Trumper, L.H., von Bonin, F., Kapp, U., Patterson, B., Snow, B.E., Mak, T.W., 2001. Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma. Blood 97, 250. Sugawara, N., Yamashita, T., Ote, Y., Miura, M., Terada, N., Kurosawa, M., 2002. TARC in allergic disease. Allergy 57, 180. Syed, F., Huang, C.C., Li, K., Liu, V., Shang, T., Amegadzie, B.Y., Griswold, D.E., Song, X.Y., Li, L., 2007. Identification of interleukin-13 related biomarkers using peripheral blood mononuclear cells. Biomarkers 12, 414. Takeda, K., Kamanaka, M., Tanaka, T., Kishimoto, T., Akira, S., 1996. Impaired IL-13-mediated functions of macrophages in STAT6deficient mice. J. Immunol. 157, 3220. Taube, C., Duez, C., Cui, Z.H., Takeda, K., Rha, Y.H., Park, J.W., Balhorn, A., Donaldson, D.D., Dakhama, A., Gelfand, E.W., 2002. The role of IL-13 in established allergic airway disease. J. Immunol. 169, 6482. Vercelli, D., 2002. Genetics of IL-13 and functional relevance of IL-13 variants. Curr. Opin. Allergy Clin. Immunol. 2, 389. Vladich, F.D., Brazille, S.M., Stern, D., Peck, M.L., Ghittoni, R., Vercelli, D., 2005. IL-13 R130Q, a common variant associated with allergy and asthma, enhances effector mechanisms essential for human allergic inflammation. J. Clin. Invest. Wang, L.M., Michieli, P., Lie, W.R., Liu, F., Lee, C.C., Minty, A., Sun, X.J., Levine, A., White, M.F., Pierce, J.H., 1995. The insulin receptor substrate-1-related 4PS substrate but not the interleukin2R gamma chain is involved in interleukin-13-mediated signal transduction. Blood 86, 4218. Wills-Karp, M., Luyimbazi, J., Xu, X., Schofield, B., Neben, T.Y., Karp, C.L., Donaldson, D.D., 1998. Interleukin-13: central mediator of allergic asthma. Science 282, 2258. Yu, B., Koga, T., Urabe, K., Moroi, Y., Maeda, S., Yanagihara, Y., Furue, M., 2002. Differential regulation of thymus- and activation-regulated chemokine induced by IL-4, IL-13, TNF-alpha and IFN-gamma in human keratinocyte and fibroblast. J. Dermatol. Sci. 30, 29. Zurawski, S.M., Vega Jr., F., Huyghe, B., Zurawski, G., 1993. Receptors for interleukin-13 and interleukin-4 are complex and share a novel component that functions in signal transduction. EMBO J. 12, 2663.
Research paper
Development of an in vitro potency bioassay for therapeutic IL-13 antagonists: The A-549 cell bioassay Renee Miller a , Ramkrishna Sadhukhan b , Chengbin Wu a,⁎ b
a Department of Biologics, Abbott Bioresearch Center, Worcester, MA 01605, United States Department of Molecular & Cell Biology, Abbott Bioresearch Center, Worcester, MA 01605, United States
Received 25 October 2007; received in revised form 13 February 2008; accepted 14 February 2008 Available online 7 March 2008
Abstract Interleukin 13 is a key cytokine that mediates airway hyper-responsiveness and mucus over-production, and several anti-IL-13 therapeutic antibodies are currently in clinical development for the treatment of asthma. Conventional cell-based bioassays for evaluating neutralization potencies of IL-13 antagonists are semi-quantitative or with a low sensitivity. Here, we report the development of a highly sensitive bioassay to assess the potency of IL-13 antagonists using human lung epithelial A-549 cells that produce thymus and activation-regulated chemokine (TARC) in response to IL-13 stimulation. The A-549 cells were responsive to both wild type and a variant form of recombinant human IL-13 in a concentration-dependent manner, with a 16 to 24 h exposure time found to be within the linear portion of the bioassay response range. The Effective Concentration at 50% of the maximal response (ED50) of IL-13 determined for the assay was 1–5 ng/mL. With this level of IL-13, an anti-IL-13 antibody B-B13 yielded an approximate median Inhibitory Concentration (IC50) value of 0.2 nM. Bioassay optimization was performed to achieve best assay condition and sensitivity. Additionally, IL-13 antagonist potency against natural human IL-13 was also investigated in the A-549 cell bioassay. © 2008 Elsevier B.V. All rights reserved. Keywords: IL-13 antagonist; IL-13 antibody; TARC ELISA (Enzyme-Linked Immunosorbent Assay); In vitro; A-549; Potency; Bioassay
1. Introduction Human IL-13 is a Th-2 cytokine that signals through its cell surface receptors via signal transducer and activator of transcription (STAT6) and insulin receptor substrate-1/2 (IRS-1/2) pathways (Wang et al., 1995; Schnyder et al., Abbreviations: IL-13, interleukin 13; TARC, thymus and activationregulated chemokine; ED50, 50%; effective concentration; IC50, median inhibitory concentration; ATCC, American Type Culture Collection; BSA, bovine serum albumin. ⁎ Corresponding author. E-mail address: [email protected] (C. Wu). 0022-1759/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.02.009
1996; Takeda et al., 1996). In recent years, IL-13 has been implicated in the pathogenesis of human asthma as elevated levels of IL-13 mRNA and protein have been detected in lungs of asthmatic patients (Huang et al., 1995), and a variant form of human IL-13, resulted from a genetic polymorphism, has been associated with asthma and atopy (Heinzmann et al., 2000; Hoerauf et al., 2002; Vercelli, 2002; Heinzmann et al., 2003; Chen et al., 2004; Vladich et al., 2005). IL-13 has been validated as a target for asthma in preclinical animal models, as anti-IL-13 antibodies reduced airway hyperresponsiveness (AHR) and mucus hyper-secretion (Grunig et al., 1998; Wills-Karp et al.,
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1998; Taube et al., 2002). Currently, several therapeutic anti-IL-13 monoclonal antibodies are in pre-clinical and clinical development for the treatment of asthma. Bioassays can provide useful information regarding the drug activity or specificity in the search for potent and effective antagonists to IL-13. Moreover, with the numerous reports of IL-13 involvement in a variety of disease states, a sensitive bioassay system for measuring the biological activity of this cytokine and its antagonists have widespread applications. Several bioassay methods for measuring the activity of IL-13 and its antagonists have been previously reported. Human erythroleukemic TF-1 cells proliferate in response to IL-13 with a low signal-tobackground ratio (~2-fold) and an ED50 N= 100 ng/mL (Zurawski et al., 1993). Other assays, such as IL-13-induced CD23 expression on human B cells (Punnonen et al., 1993) and IL-13-induced expression of 15-lipoxygenase in human blood monocytes (Roy et al., 2002) are not quantitative, therefore cannot be used for precise measurements of potency of high affinity anti-IL-13 therapeutic monoclonal antibodies. IL-13 was previously reported to induce TARC production by the human lung epithelial adenocarcinoma cell line, A-549, in the presence of recombinant human TNF-alpha (rhTNF-α)(Yu et al., 2002; Faffe et al., 2003). This observation formed the basis of the bioassay discussed here, which provides several advantages over some of the bioassays previously mentioned. In addition, we also demonstrate the application of this assay in evaluating antibody potency against natural IL-13 secreted from human cells. Since IL-13 and IL-4 can signal through a common receptor system IL-13Rα1/IL-4R, and they are also co-produced/co-exist in many biological systems, it has been difficult to generate natural IL-13 without IL-4 also being produced simultaneously. Here we show that biologically active IL-13, but not IL-4, is produced from human PBMC under specific stimulation conditions, which could be applied to evaluating neutralization activity of therapeutic IL-13 antagonists. 2. Materials and methods Recombinant human IL-2, human IL-13 (wild type), mouse IL-13, rat IL-13, rhesus IL-13, anti-IL-13Rα1 polyclonal antibody (AF152), anti-13Rα2 polyclonal antibody (AF146), IL-13Rα1/Fc, IL-13Rα2/Fc fusion proteins (SF21 and NS0-derived), and human cytokine (IL-13, IL-4, and TARC) ELISA kits were purchased from R&D Systems. Anti-IL-13 monoclonal antibody clone B-B13 was purchased from Diaclone. Human variant IL-13 was purchased from PeproTech. Sheep and cynomolgus monkey IL-13 proteins were generated from
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transiently transfected COS-7 cells. A-549 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and were cultured in complete F12 growth medium (Gibco), containing 10% fetal bovine serum and supplemented with the 1% L-glutamine, 1% sodium bicarbonate, 50 units/mL penicillin, and 50 μg/mL Streptomycin. Mononuclear cell enriched leukocyte packs were obtained from Biological Specialty Corporation (Colmar, PA). 2.1. Optimizing the concentration of rhTNF-α for the A-549 bioassay A-549 cells were seeded 1 day prior to the experiment at 4 × 105 and 1 × 105 cells per well densities in a 96-well culture plate. On the day of the experiment, medium was replaced with100 μl fresh medium containing rhTNF-α (6.25 to 3.200 ng /mL). Additionally, rhIL-13 was also added to cells at a final concentration of 5 ng/mL. All well volumes were adjusted to 200 μL. After a 16–20 h overnight incubation, the supernatants were collected and TARC levels determined by ELISA. The optimum rhTNF-α concentration was determined by calculating the average TARC values, which were processed by GraphPad Prism. 2.2. Determining the A-549 cell density A-549 cells were plated 1 day prior to the experiment at four different seeding densities: 4 × 105, 2 × 105, 1 × 105 and 0.5 × 105 cells per well in 96-well culture plates. On the day of the experiment, medium was replaced with100 μl fresh medium containing rhTNF-α (the final concentration of rhTNF-α was 200 ng/mL). Additionally, rhIL-13 was also added to cells at a final concentration of 5 ng/mL. All well volumes were adjusted to 200 μL. After a 16–20 h overnight incubation, the supernatants were collected and TARC levels determined by ELISA. The optimum rhTNF-α concentration was determined by calculating the average TARC values, which were processed by GraphPad Prism. 2.3. Biological activity of wild type and variant rhIL-13, as well as IL-13 of other species in the A-549 bioassay A-549 cells were plated 1 day prior to the experiment at 2 × 105 cells per well. On the day of the experiment, medium was replaced with100 μl fresh medium containing rhTNF-α (the final concentration of rhTNF-α was 200 ng/mL). Subsequently, recombinant human (wt and variant), mouse, rat, sheep, rhesus and cynomolgus monkey IL-13 in fresh complete medium was added to
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media, and plated into 4 × 150 mm plate (40 ml/plate). In two plates, 400 ng recombinant human IL-4 (rhIL-4) and 200 µg anti human IL-12 (MAB219, R&D Systems) were added to induce a Th2 condition and marked as “skewed”. The other two plates were without rhIL-4 and anti-IL-12 and labeled as “unskewed”. The cells were cultured for 3 days. On day 4, cells from each individual plate were collected and resuspended in 40 ml complete media containing 10 ng/ml recombinant human IL-2, followed by incubating for additional 7 days to allow the cells to expand. On day 8, cells were counted and seeded into 24-well plate at 1.6 × 106 cells/well in 1 ml of complete media, then activated to produce cytokines (IL-13) by treatment with or without PMA and ionomycin. After 48 h, the IL-13-containing supernatants were harvested and cytokine levels determined by ELISA. The conditional media containing natural IL-13 were able to induce TARC in A-549 bioassay, and were further analyzed for antibody inhibition.
Fig. 1. Development and optimization of A-549 bioassay. A, Recombinant hTNF-α was titrated in the presence of rhIL-13 (5 ng/ mL) at 2 different cell densities, 1 ×105/well (○) and 4×105/well (■) (n=4, ⁎pb 0.01 between the 2 cell density groups). B, A-549 cells were further titrated at four different seeding densities: 4×105, 2×105, 1×105 and 0.5×105 cells/well in the presence of rhTNF-α (200 ng/mL) and rhIL13 (5 ng/mL). TARC production was measured by ELISA after a 16-h stimulation (n=4, ⁎pb 0.01 compared to the lowest cell density group). Data shown are representative of 3 independent experiments, and each data point was represented by quadruple wells.
each well, with the final concentration of IL-13 ranged from 0.015 to1000 ng/mL. After a 16–20 h overnight incubation, the supernatants were harvested and TARC levels determined by ELISA. EC50 values were calculated by GraphPad Prism software. 2.4. Generation of natural human IL-13 from PHA blast cells Human PHA Blasts were generated from lymphocytes (isolated from mononuclear cell enriched leukocyte packs) as previously described (Keen et al., 2006). PHA-blasted cells (1 × 108 cells/vial) were quickly thawed and mixed together, washed once with RPMI complete media, resuspended in 160 ml of complete
Fig. 2. IL-13 induces TARC production by A-549 cells in the presence of TNF-α. A, Recombinant wt (■) and variant (○) hIL-13 were titrated in the presence of rhTNF-α (200 ng/mL) at 2 × 105/well cell density. B, IL-13 of other species, including rat IL-13 (□), mouse IL-13 (▼), sheep IL-13 (▲), rhesus monkey IL-13 ( ), and cynomolgus monkey IL-13 ( ), can also induce TARC production, with potencies similar to human IL-13. TARC production was measured by ELISA after a 16-h stimulation. Data shown are representative of 3 independent experiments, and each data point was represented by quadruple wells (n = 4).
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◇
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2.5. Antibody Neutralization of recombinant and natural hIL-13 A-549 cells were plated 1 day prior to the experiment at 2 × 105 cells per well. On the day of the experiment, medium was replaced with100 μl fresh medium containing rhTNF-α (the final concentration of rhTNF-α was 200 ng/mL). The plates were placed at 37 °C, 5% CO2 until addition of mixture of human IL-13 (purified recombinant human IL-13 or nature IL-13 conditional medium) and IL-13 antagonists. Human IL-13 in complete F12 media (50 μL at 20 ng/mL) was mixed with IL-13 antagonists (50 μL from 1 pM to 5 μM), and the mixture was added to the rhTNF-α treated A-549 cells. All well volumes were equal to 200 μL. The final concentration of human IL-13 was 5 ng/mL. After a 16– 20 h incubation, the supernatants were harvested and TARC levels determined by ELISA. Neutralization potencies of IL-13 antagonists were calculated based on the average TARC values for each data point and the IC50 values determined by GraphPad Prism software.
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2.6. Statistical analysis ANOVA with post hoc Bonferroni correction was used to perform statistical analysis. Data are expressed as the mean ± SEM and a p b 0.05 was considered to indicate significant differences. 3. Results 3.1. Developing A-549 bioassay Several important assay parameters were optimized to establish an IL-13 bioassay with a high sensitivity. Recombinant hTNF-α was titrated (1600 ng/mL– 6.25 ng/mL) in the presence of a fixed amount of rhIL13 (5 ng/mL) and at different A-549 seeding cell densities. The final concentration of IL-13 at 5 ng/mL was determined from earlier work on IL-13 titration in the presence of several different hTNF-α concentrations (IL-13 ED50 ~ 1–5 ng/mL). Under these conditions, 200 ng/mL hTNF-α induced the maximum TARC
Fig. 3. Bioactive natural human IL-13 production from PHA-P blast cells. Human PBMC cells were PHA-P treated and incubated with or without PMA or ionomycin, under Th2-skewed (black bars) or unskewed (hatched bars) conditions, followed by expansion in the presence or absence of rhIL-2. Natural human IL-13 (A) and IL-4 (B) productions were measured by ELISA after a 48-h expansion. Data shown are pooled from 3 independent experiments using cells from 2 different donors, and each data point was represented by quadruple wells. There was no statistical difference between skewed and unskewed conditions for all groups except two (indicated by ⁎p b 0.01). The bioactivity of natural IL-13 was analyzed in the A-549 bioassay in comparison to E. coli-derived recombinant human wt IL-13 (both at 5 ng/mL) (C). Both induced similar amount of TARC without statistical difference.
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production by the A-549 cells, as additional rhTNF-α did not further increase the overall TARC signal (a representative experiment is shown in Fig. 1A). Subsequently, A-549 cell densities were titrated at 4 × 105, 2 × 105, 1 × 105 and 0.5 × 105 cells per well in the presence of 200 ng/mL rhTNF-α and 5 ng/mL hIL-13. As shown in Fig. 1B, cell density at 2 × 105 cells per well produced the maximum TARC levels, and there was no further increase in TARC production by increasing the cell number from 2 × 105 to 4 × 105 cells/well. Lastly, recombinant wt and variant IL-13 was titrated (1000 ng/mL–0.15 ng/mL) in the presence of a fixed amount of rhTNF-α at 200 ng/mL and cell density at 2 × 105 cells per well. TARC production increased in response to increasing concentrations of IL-13, with ED50 values in the range of 1-5 ng/mL for both wt and variant IL-13 (Fig. 2A). Consequently, hIL-13 concentration of 5 ng/mL was selected for subsequent neutralization assays. In addition, A-549 cells also responded to mouse, rat, sheep, cynomolgus, and rhesus IL-13, with ED50 values similar to that of human IL-13 (Fig. 2B). Culture supernatants from A-549 cells treated with medium alone or medium with 200 ng/mL rhTNF-α did not induce detectable levels of TARC. A-549 cells up to passage #55 (post acquisition from ATCC) have been used in this assay with consistent signal of response and reproducibility.
did not produce high levels of IL-13 (Fig. 3A), and subsequently did not induce detectable levels of TARC in the A-549 bioassay. 3.3. Determining neutralization potency of recombinant human IL-13 (rhIL-13) antagonists IL-13 antagonists were titrated (20 μg/mL–0.0003 μg/mL) in the presence of a fixed amount of rhTNF-α (200 ng/mL) and hIL-13 (5 ng/mL). Under these conditions, the concentration-dependent inhibition of TARC production by various IL-13 antagonists is shown in Fig. 4. Anti-IL-13 monoclonal antibody B-B13 and IL-13Rα2/Fc (SF21 cell-derived) exhibited high potencies in inhibiting IL-13-induced TARC production, with IC50 values of 0.2 and 0.7 nM, respectively. Interestingly, NS0-derived IL-13Rα2/Fc was less potent than that produced by SF21 cells. Both IL-13Rα1/Fc fusion protein and an anti-IL-13Rα1 polyclonal antibody showed low potencies in this assay (IC50 N 10 nM). In addition, an antiIL-13Rα2 polyclonal antibody did not show any inhibitive activity. Collectively, these data indicate that IL-13-induced TARC production by A-549 cells is mediated through IL-13Rα1/IL-4 receptor complex. This is consistent with the observation that IL-4 could also induce TARC production by A-549 cells concentration-dependently in the presence of TNF-α, with an ED50
3.2. Natural human IL-13 production from PHA blast cells Natural IL-13 was produced by using mitogen (PHA) activated human peripheral mononuclear cells. No significant difference in production of IL-13 was observed between Th2-skewed and unskewed conditions(Fig. 3A). PMA stimulation of IL-2-treated cells induced 10–15 ng/mL IL-13 production, and the presence of ionomycin did not further enhance IL-13 levels (Fig. 3A). In addition, the presence of ionomycin induced high levels of IL-4 under both Th2-skewed and unskewed conditions (Fig. 3B), indicating that activation of the PKC pathway was sufficient for IL-13 secretion, whereas activation of calcium signaling pathways was required for IL-4 production. There was no significant production of TARC under any of the conditions tested (data not shown). Therefore, in the absence of ionomycin, the lack of production of both IL-4 and TARC rendered the natural IL-13 produced suitable for testing in the A-549 bioassay. The natural IL-13 produced by this method at 5 ng/ml was able to induce TARC production by A-549 cells to similar levels as recombinant IL-13 (Fig. 3C). Culture supernatants from PHA Blasts treated with medium alone or PMA without IL-2
Fig. 4. Neutralization of rhIL-13-induced TARC production by IL-13 antagonists. Various IL-13 antagonists were titrated in the presence of recombinant human wt IL-13 (5 ng/mL) and rhTNF-α (200 ng/mL) at 2 × 105/well A-549 cell density. Antagonists included anti-IL-13 mAb B-B13 (■), rhIL-13Rα2/Fc (Sf21-derived) (△), rhIL-13Rα2/Fc (NS0-derived) ( ), anti-hIL-13Rα1 pAb (□), rhIL13Rα1/Fc (○), anti-hIL-13Rα2 pAb (▽), and a non-specific control mouse IgG (●). TARC production was measured by ELISA after a 16-h stimulation. Data shown are representative of 3 independent experiments, and each data point was represented by quadruple wells.
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Fig. 5. Neutralization of natural human IL-13-induced TARC production by IL-13 antagonists. IL-13 antagonists were titrated in the presence of natural human IL-13 (5 ng/mL) and rhTNF-α (200 ng/mL) at 2 × 105/well A-549 cell density. Antagonists included anti-IL-13 mAb B-B13 (■) and rhIL-13Rα2/Fc (□), and a non-specific control mouse IgG (●) is also included. TARC production was measured by ELISA after a 16-h stimulation. Data shown are representative of 3 independent experiments, and each data point was represented by quadruple wells.
of 0.1 nM (data not shown). In addition, anti-IL-13 mAb B-B13 and IL-13Rα2/Fc were able to neutralize naturally derived human IL-13 in a concentration-dependent manner (Fig. 5). The neutralization potencies (IC50 = 0.4 and 1.1 nM for B-B13 and IL-13Rα2, respectively) were similar to that observed against E-coli-derived recombinant human IL-13 for both antagonists. Furthermore, both antagonists were able to inhibit TARC production to 100%, indicating all TARC production was due to the activity of natural IL-13, and there was no IL-4 contamination. By RT-PCR and DNA sequencing, the IL-13 produced by this particular donor was also determined to be wt IL-13. 4. Discussion A robust bioassay is instrumental for the selection and development of a therapeutic monoclonal antibody. This report demonstrates a new bioassay with a desirable sensitivity that can support selection and pre-clinical development of a therapeutic IL-13 antagonist. Several methods for measuring IL-13 and its antagonist activity have been described previously (Punnonen et al., 1993; Zurawski et al., 1993; Roy et al., 2002). These methods possess some common limitations, such as the requirement for high IL-13 concentrations, low signal-to-background ratio, even with radioactive endpoints. Therefore, a novel bioassay was developed in our lab to assess in vitro potency of IL-13 and IL-13 antagonists, which exhibited some advantages over previously described methods. The A-549 cell line was shown previously to be an IL-13 responsive cell line, releasing TARC upon
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exposure to IL-13 in the presence of TNF-α (Yu et al., 2002; Faffe et al., 2003). Building on this observation, we established a reproducible bioassay method with a simple endpoint for measuring potency of IL-13 antagonists. The A-549 cell potency assay provides a valuable tool for assessing neutralizing activity of various IL-13 antagonists, without the use of [3H]-thymidine or special laboratory equipment and reagents. The A-549 bioassay is based on the direct measurement of IL-13 induced TARC from A-549 cell supernatants using a standard commercial ELISA, therefore it can be performed routinely in most laboratories. The endpoint for the A-549 assay is quantitative, and the A-549 cells have been found to respond to IL-13 consistently over many passages. A-549 cells also respond to IL-13 of different species with similar sensitivity (similar ED50), including human (wt and variant), cynomolgus and rhesus monkey, mouse, rat, and sheep IL-13. This feature of the bioassay is important to enable analyzing species cross-reactivity of any therapeutic IL-13 antagonists for toxicological studies. In contrast, the conventional B9 cell-based bioassay responds to human IL-13 approximately 100fold less sensitively than to mouse IL-13 (Lauder and McKenzie, 2001). Most therapeutic antibodies are generated against recombinant target proteins, either by in vivo hybridoma technology or in vitro phage display technology. As various types of differences exist between recombinant and naturally derived proteins (i.e. structure, glycosylation, activity, etc), it is critical to assess whether the therapeutic antibody can also recognize naturally derived target protein equally well. To this end, we established an in vitro system to produce natural human IL-13. However, since A-549 cells also respond to IL-4 and perhaps other mediators, it was therefore critical to induce IL-13 production without the induction of IL-4 secretion. This would significantly simplify the assay, as no further purification step to remove the IL-4 would be required. However, producing high levels of natural IL-13 without IL-4 secretion has proven difficult, as these two cytokines are often co-produced in many biological systems, i.e. a Th2-polarized T cell cultural condition. In this report, natural IL-13 was produced by pre-activated human PBMCs in the presence of PMA, ionomycin, or in combination, followed by IL-2 stimulation, in both Th2skewed and unskewed conditions. Interestingly, IL-4 production required the presence of ionomycin, followed by IL-2 stimulation, in both Th2-skewed and unskewed conditions. This indicated that activation of calcium signaling and subsequent activation of protein kinase C was required for IL-4 production but not for IL-13 production in this particular system. Therefore, we have
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developed an in vitro system for producing natural IL-13 without IL-4 production. In addition, the concentrations of natural IL-13 produced in this system was much higher than those produced by previously described Hodgkin lymphoma cell lines, such as HDLM-2 (Skinnider et al., 2001). Furthermore, we have determined that HDLM-2 cells spontaneously released high concentrations of TARC (N 20 ng/106 cells/ml/day), therefore natural IL-13 produced by HDLM-2 cells could not be readily used for A-549 bioassay prior to removal of existing TARC. In contrast, our current system does not product TARC under any of the conditions tested. Consequently, complete inhibition of TARC was achieved in natural IL-13 neutralization assay with both an anti-IL-13 mAb (B-B13) and IL-13Rα2/Fc fusion protein, indicating all TARC had been produced by the A-549 cells in response to natural IL-13. We demonstrate here that the IL-13 induces TARC expression in a lung epithelial adenocarcinoma cells in vitro. Alone, neither IL-13 nor TNF-α was effective. The combination of IL-13 and TNF-α synergistically stimulated TARC production. Synergy between IL-13 and TNF-α has previously been demonstrated for the induction of TARC from human peripheral blood mononuclear cells (Nomura et al., 2002), human keratinocyte and fibroblast (Yu et al., 2002), human bronchial epithelial cells (Sekiya et al., 2000), and human airway smooth muscle cells (Faffe et al., 2003). Similar synergistic effect was also observed for eotaxin release from airway epithelial and smooth muscle cells (Matsukura et al., 2001; Moore et al., 2002). Our study indicated that TARC production from the A-549 cells was the result of the induction of TARC expression, and not due to the proliferation of A-549 cells. Although IL-13 could induce minor proliferation for certain lung epithelial cells (Booth et al., 2001), we in fact did not detect any significant increase in A-549 cell numbers after incubating with IL-13 and TNF-α under the current assay condition (data not shown). It has been suggested that the synergy between TNF-α and IL-13 on eotaxin release could be transcriptionally mediated, as the STAT-6-binding element in the eotaxin promoter overlaps with a consensus binding site for nuclear factor (NF)-κB, and that simultaneous binding of both STAT-6 and NF-κB to this region of the eotaxin promoter results in enhanced promoter activity (Matsukura et al., 1999). As CCchemokine (including both TARC and eotaxin) genes are clustered within the same chromosomal region, we speculate that IL-13/ TNF-α synergy in TARC induction could be similar to eotaxin induction. As TARC has been implicated in asthma (Kawasaki et al., 2001; Berin, 2002; Sugawara et al., 2002), and has been suggested to be a
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