Human studies using Abs to two different, nonoverlapping epitopes of IL-13 suggested that epitope specificity can have a clinically significant impact on clearance of IL-13. We propose that Ab modulation of IL-13 interaction with IL-13Rα2 underlies this effect. Two Abs were administered to healthy subjects and mild asthmatics in separate dose-ranging studies and allergen-challenge studies. IMA-638 allows IL-13 interaction with IL-13Rα1 or IL-13Rα2 but blocks recruitment of IL-4Rα to the IL-13/IL-13Rα1 complex, whereas IMA-026 competes with IL-13 interaction with IL-13Rα1 and IL-13Rα2. We found ∼10-fold higher circulating titer of captured IL-13 in subjects treated with IMA-026 compared with those administered IMA-638. To understand how this difference could be related to epitope, we asked whether either Ab affects IL-13 internalization through cell surface IL-13Rα2. Humans inducibly express cell surface IL-13Rα2 but lack the soluble form that regulates IL-13 responses in mice. Cells with high IL-13Rα2 expression rapidly and efficiently depleted extracellular IL-13, and this activity persisted in the presence of IMA-638 but not IMA-026. The potency and efficiency of this clearance pathway suggest that cell surface IL-13Rα2 acts as a scavenger for IL-13. These findings could have important implications for the design and characterization of IL-13 antagonists.

Interleukin-13 neutralization has the potential to treat asthma and other inflammatory diseases (1). We developed two Abs to IL-13, which block responses mediated through the IL-13Rα1/IL-4Rα complex (2). IMA-026 inhibits the initial interaction between IL-13 and IL-13Rα1, whereas IMA-638 allows IL-13 interaction with IL-13Rα1 but blocks recruitment of the IL-4Rα–chain to form the high-affinity receptor. Both IMA-638 and IMA-026 have been administered to humans in separate dose-ranging studies, as well as in allergen-challenge studies in mild asthmatics (3). Interestingly, subjects treated with IMA-026 displayed higher concentrations of serum IL-13 than did those given IMA-638. We asked whether the distinct IL-13 epitope recognition by these Abs could have contributed to the observed differences in circulating IL-13 concentration. Our findings support a model in which cell surface IL-13Rα2 efficiently depletes extracellular IL-13, a process that is efficiently antagonized by IMA-026 but not by IMA-638. The differences in circulating IL-13 concentration seen in human studies with these Abs suggest that IL-13Rα2 is a critical physiological regulator of IL-13 clearance.

IL-13Rα1 and IL-13Rα2 recognize overlapping epitopes of IL-13, but IL-13Rα2 interacts with a larger binding interface, which helps to account for its unusually high affinity and competitive advantage for binding IL-13 (4). Described primarily as a decoy, IL-13Rα2 is a potent antagonist of IL-13 bioactivity mediated through the IL-13Rα1/IL-4Rα complex on human fibroblasts (58), epithelial cells (911), keratinocytes (12, 13), and smooth muscle cells (12, 14, 15). Recent reports suggested that IL-13Rα2 may have some signaling capacity, resulting in fibrotic responses to IL-13 (16). The receptor itself has only 17 aa in the cytoplasmic domain (17), but it may be able to interface with additional signaling components to activate AP-1 (16, 18), MAPK (9), ERK-1/2 (19, 20), or STAT3 (21) pathways. No direct interaction of this receptor with signaling molecules has been demonstrated, and there is no clear consensus on the signaling pathways affected by engagement of IL-13Rα2.

In the mouse, a soluble form of IL-13Rα2 can be readily detected in circulation and plays an important role in regulation of IL-13 bioactivity. Mice lacking IL-13Rα2 have high levels of IL-13 in tissues and display functional responses consistent with overexpression of IL-13, as would be expected from lack of decoy activity (2224). They also have reduced titers of circulating IL-13 (22), suggesting that the soluble form of IL-13Rα2 could act as a carrier or chaperone for IL-13 in the circulation. The mouse soluble IL-13Rα2 is most likely generated by an alternatively spliced form of the transcript encoding cell surface IL-13Rα2 (25). A counterpart to this alternatively spliced transcript has not been described in humans, and soluble IL-13Rα2 has not been detected in human serum or bronchoalveolar lavage fluid (26). This raises the possibility that regulation of IL-13 bioactivity through the action of IL-13Rα2 is quite distinct between rodents and humans.

In humans, cell surface expression of IL-13Rα2 is highly regulated, and it is inducible on fibroblasts, smooth muscle cells, keratinocytes, and other cell types following exposure to IL-13, IL-4, TNF-α, IFN-γ, or combinations of these cytokines (7, 14, 27). IL-13Rα2 efficiently mediates internalization of bound cytokine (28), and it was suggested that IL-13Rα2 mediates clearance of local IL-13 (29), although this has not been clearly demonstrated in vivo. With inducible expression on several cell types that make up the bulk of tissue, cell surface IL-13Rα2 could be a critical modulator of IL-13 exposure. We provide evidence in support of this concept from human studies, which suggest that IL-13Rα2 could function as a major regulator of IL-13 exposure and clearance.

Ab to human IL-13 was generated in BALB/c mice and humanized at Pfizer Research to generate IMA-638 (IgG1, κ). A second Ab was raised in BALB/c mice against peptides corresponding to the amino acid sequence of cynomolgus monkey IL-13 (30), conjugated to keyhole limpet hemocyanin, and humanized at Pfizer Research to generate IMA-026 (IgG1, κ). Carimune NH immune globulin i.v. (human intravenous immunoglobulin [IVIG]; ZLB Bioplasma, Bern, Switzerland) was depleted of non-Ig components by passage over a protein A column, for use as control Ab. Recombinant human IL-4–FLAG and human IL-13–FLAG were generated at Pfizer Research.

IMA-638 and IMA-026 were evaluated in separate single ascending-dose studies. IMA-638 was administered to mild-moderate asthmatics at doses of 0.3, 1, 2, and 4 mg/kg s.c. with placebo control, as well as 3 mg/kg i.v. IMA-026 was administered to healthy subjects at doses of 0.3, 1, 2, and 4 mg/kg s.c. with placebo control, as well as 3 mg/kg i.v. Groups of 7–10 subjects received each dose. IMA-638 and IMA-026 were also tested in separate double-blind, randomized, placebo-controlled allergen-challenge studies in stable mild asthmatics, as described (3). Groups of 14–15 subjects each received two s.c. doses of humanized IL-13 Ab, 2 mg/kg each, administered 1 wk apart. All studies were approved by the ethics research boards of the respective institutions as decribed (3), and signed informed consent was obtained from all subjects. Concentrations of anti–IL-13 Ab and IL-13 cytokine in serum samples were determined as described (3).

HT-29 or A375 cells were plated in microtiter wells and grown to ∼70% confluence. HT-29 cells were cultured in a 5% CO2 incubator in McCoy’s 5A medium containing 10% FCS, 0.15% sodium bicarbonate, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. A375 cells were cultured in a 10% CO2 incubator in DMEM containing 10% FCS, 10 mM HEPES, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Recombinant human IL-13 was added in the presence or absence of Abs IMA-638, IMA-026, or control human Ig (IVIG) and incubated for the indicated time period at 37°C. Supernatants were harvested, and the residual IL-13 in the medium was quantitated.

Separate assays were developed to detect total IL-13, with no interference from IMA-638 or IMA-026. Both free and Ab-bound IL-13 were detected by these assays. To detect IL-13 in the presence or absence of IMA-638, ELISA plates (Nunc Maxi-Sorp) were coated with mouse anti–IL-13 Ab (Jin2; Pfizer Research), washed with PBS containing 0.05% Tween-20, and then blocked with 0.5% gelatin in PBS. Recombinant human IL-13 standard (R&D Systems) or dilutions of cell culture supernatant was added in PBS containing 0.05% Tween-20 containing 2% FCS. After 4 h, plates were washed, and biotinylated mouse anti–IL-13 Ab (Ab64; Pfizer Research), was added for 1–2 h, followed by HRP-streptavidin (Southern Biotechnology Associates, Birmingham, AL). Color was developed using Sure Blue peroxidase substrate (KPL, Gaithersburg, MD). Absorbance was read at 450 nm (SpectraMax; Molecular Devices, Sunnyvale, CA). Detection of IMA-026 in the presence or absence of IL-13 was performed in a similar manner, except that plates were coated with mouse anti–IL-13 Ab (mAb13.2; Pfizer Research).

A375 or HT-29 cells were used as targets cells. The cells were washed into RPMI 1640 containing 10% FCS and incubated for 1 h, 37°C, with 100 μCi [51Cr] (PerkinElmer Life Sciences, Boston, MA) in a minimal volume of media. The cells were washed in serum-free RPMI 1640, incubated for 1 h at room temperature to allow [51Cr] to “bleed” from the cells, washed, and plated in RPMI 1640 containing 10% FCS in 96-well round-bottom plates at 1 × 104/well. Recombinant human IL-13 was added at 10 nM and preincubated for 10 min before addition of effector cells. Effector cells were prepared from fresh blood drawn from healthy volunteers into heparinized Vacutainer tubes (Becton-Dickinson). NK cells were enriched using Rosette Sep human NK cell enrichment mixture (StemCell Technologies, Vancouver, BC, Canada). Cells were washed into RPMI 1640 containing 10% FCS, added to the target cells at an E:T cell ratio of 8:1, and incubated for 5 h in a 5% CO2 incubator at 37°C with the indicated concentration of Ab. Control wells received no Ab (background) or 100 μl 0.1% Triton X-100 (total release). Supernatants were harvested using a Skatron harvesting press (Molecular Devices-Skatron, Sunnyvale, CA), and [51Cr] release was determined by counting in a Wallac Wizard gamma counter (PerkinElmer).

HT-29 or A375 cells (both from American Type Culture Collection, Manassas, VA) were washed in ice-cold PBS containing 1% BSA and 0.001% sodium azide. Cells were incubated with 20 ng/ml human IL-13 for 20 min at 4°C, washed, and incubated for an additional 20 min at 4°C with biotinylated IMA-638, IMA-026, or control human Ig. Alternatively, untreated cells were incubated with Abs to human IL-13Rα1 (R&D Systems) or IL-13Rα2 (R&D Systems). The cells were stained with PE-labeled streptavidin. For the STAT6-phosphorylation assay, HT-29 cells were incubated with 1 ng/ml human IL-13 for 30 min at 37°C, fixed in 1% paraformaldehyde, treated overnight at −20°C in absolute methanol, and stained with Alexa Fluor 488-labeled Ab to STAT6 (BD Biosciences). Fluorescence was analyzed with a FACSCalibur (BD Biosciences).

A375 cells were cultured on microscope chamber slides (BD Biocoat) for 24 h at 37°C with recombinant human IL-4–FLAG or IL-13–FLAG in the presence or absence of biotinylated IMA-638, IMA-026, or control human Ig. For cell surface staining, cells were washed in PBS containing 1% BSA and incubated with Cy3 anti-FLAG (Sigma) and Alexa Fluor 488-streptavidin (Invitrogen) for 30 min at 4°C. For intracellular staining, cells were washed, fixed with Cytofix-Cytoperm (BD Biosciences), and incubated with Cy3 anti-FLAG (Sigma), Alexa Fluor 488-streptavidin (Invitrogen), or Alexa Fluor 488-phalloidin (Invitrogen), and TO-PRO 3 (Molecular Probes). Cells were fixed in 1% paraformaldehyde, rinsed well with water, and dried. Cover slips were mounted using Pro-Long (Molecular Probes). Slides were read using a Nikon TE-300 microscope with Bio-Rad Radiance 2000 confocal system (Carl Zeiss Microimaging, Thornwood, NY).

We developed two high-affinity neutralizing Abs to human IL-13, IMA-638 and IMA-026, which bind to distinct, nonoverlapping epitopes (2). IMA-026 blocks IL-13 interaction with IL-13Rα1. IMA-638 permits IL-13 interaction with IL-13Rα1, but it blocks recruitment of IL-4Rα to the complex (2). Both Abs are highly efficient antagonists of IL-13 bioactivity in numerous assay formats in vitro, and both showed neutralization activity in a cynomolgus monkey model of Ascaris-induced lung inflammation (2). These Abs were tested in human single-ascending dose studies and in an allergen-challenge model. Both Abs displayed half-lives of 28–30 d, with 70–80% bioavailability after s.c. dosing and reached similar serum concentrations post-s.c. administration (Fig. 1) (3).

FIGURE 1.

Serum concentration of anti–IL-13 Ab in human dose-ranging studies. Anti–IL-13 Abs were administered at the indicated doses and routes. A, IMA-638 in mild asthmatic subjects. B, IMA-026 in healthy volunteers. Serum concentrations of IMA-638 or IMA-026 were determined by ELISA.

FIGURE 1.

Serum concentration of anti–IL-13 Ab in human dose-ranging studies. Anti–IL-13 Abs were administered at the indicated doses and routes. A, IMA-638 in mild asthmatic subjects. B, IMA-026 in healthy volunteers. Serum concentrations of IMA-638 or IMA-026 were determined by ELISA.

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A highly sensitive assay was established to quantitate IL-13 in the circulation (31). The assay was modified to detect IL-13 in the presence of either IMA-638 or IMA-026 and, thus, allowed determination of total (free + Ab-bound) IL-13. Serum IL-13 titer following IMA-638 administration was quantitated in dose-ranging studies in mild asthmatic subjects (Fig. 2A) and in an allergen-challenge study in mild asthmatic subjects (3). Serum IL-13 titer following IMA-026 administration was quantitated in dose-ranging studies in healthy subjects (Fig. 2B) and in a separate allergen-challenge study in mild asthmatic subjects (3). Data from these studies revealed ∼10-fold higher serum IL-13 titer (free and Ab-bound IL-13) following dosing with IMA-026 compared with IMA-638.

FIGURE 2.

Serum concentration of IL-13 cytokine in human dose-ranging studies. Serum IL-13 concentration was determined using a highly sensitive Singulex assay. The mean baseline IL-13 concentration was 0.08–0.10 pg/ml. A, IMA-638 in mild asthmatic subjects. B, IMA-026 in healthy volunteers.

FIGURE 2.

Serum concentration of IL-13 cytokine in human dose-ranging studies. Serum IL-13 concentration was determined using a highly sensitive Singulex assay. The mean baseline IL-13 concentration was 0.08–0.10 pg/ml. A, IMA-638 in mild asthmatic subjects. B, IMA-026 in healthy volunteers.

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The human colonic epithelial line, HT-29, expresses IL-13Rα1 and IL-4Rα (32) but not IL-13Rα2, whereas the human melanoma line, A375, expresses IL-13Rα2 but not IL-13Rα1 (Fig. 3A). Incubation with IL-4 or IL-13 has been shown to induce IL-13Rα2 on primary cells (33). IL-13 increased IL-13Rα2 cell surface expression on A375 cells but IL-4 had no effect (Fig. 3B), consistent with the lack of IL-4 responsiveness by these cells. To compare uptake of extracellular IL-13 by the HT-29 and A375 lines, cells were incubated with 0.8 ng/ml IL-13 for up to 48 h at 37°C. At various time points, the concentration of residual IL-13 in the culture medium was assayed by ELISA. Although IL-13 induced STAT6 phosphorylation in HT-29 cells (Fig. 3C), indicating a functional response through the IL-13Rα1/IL-4Rα complex, IL-13 was not efficiently depleted by these cells (Fig. 3D). In contrast, IL-13 was rapidly depleted in the presence of IL-13Rα2–expressing A375 cells (Fig. 3E). By 24 h of culture, no residual IL-13 could be detected in the A375 extracellular medium. Fifty percent depletion of relatively low (0.8 ng/ml) and high (18 ng/ml) IL-13 concentrations required ∼4 and 26 h, respectively (Fig. 3E, 3F).

FIGURE 3.

IL-13R expression by cell lines and depletion of extracellular IL-13 by cells expressing IL-13Rα2. A, Expression of IL-13Rα1 and IL-13Rα2 on the human colonic epithelial line (HT-29) and the human melanoma line (A375), as determined by flow cytometry. B, A375 cells were incubated overnight with no cytokine (untreated) or with 25 ng/ml human IL-4 or IL-13. Cells were stained with biotinylated Ab to human IL-13Rα1 or IL-13Rα2, followed by PE-streptavidin. The geometric mean intensity of IL-13Ra2 expression is indicated. C, STAT6 phosphorylation by HT-29 cells was determined by flow cytometry, following 30 min of exposure to the indicated concentration of IL-13. Uptake of extracellular IL-13 by HT-29 cells with 0.8 ng/ml IL-13 (D), A375 cells with 0.8 ng/ml IL-13 (E), or A375 cells with 18 ng/ml IL-13 (F). Cells were incubated with IL-13 at 37°C. At the indicated time points, the concentration of residual IL-13 in the culture medium was assayed by ELISA.

FIGURE 3.

IL-13R expression by cell lines and depletion of extracellular IL-13 by cells expressing IL-13Rα2. A, Expression of IL-13Rα1 and IL-13Rα2 on the human colonic epithelial line (HT-29) and the human melanoma line (A375), as determined by flow cytometry. B, A375 cells were incubated overnight with no cytokine (untreated) or with 25 ng/ml human IL-4 or IL-13. Cells were stained with biotinylated Ab to human IL-13Rα1 or IL-13Rα2, followed by PE-streptavidin. The geometric mean intensity of IL-13Ra2 expression is indicated. C, STAT6 phosphorylation by HT-29 cells was determined by flow cytometry, following 30 min of exposure to the indicated concentration of IL-13. Uptake of extracellular IL-13 by HT-29 cells with 0.8 ng/ml IL-13 (D), A375 cells with 0.8 ng/ml IL-13 (E), or A375 cells with 18 ng/ml IL-13 (F). Cells were incubated with IL-13 at 37°C. At the indicated time points, the concentration of residual IL-13 in the culture medium was assayed by ELISA.

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Flow cytometry confirmed that in the presence of IL-13, IMA-638 could label IL-13Rα2–expressing A375 cells (Fig. 4). This staining was not seen in the presence of IL-4 or in the absence of cytokine (data not shown). Neither IMA-026 nor control Ig bound to the cells under any conditions (Fig. 4). As the IL-13 concentration was increased, the amount of bound IMA-638 increased (Fig. 4). These observations indicated that IMA-638 does not obscure the IL-13Rα2–binding epitope of IL-13, such that IL-13 could bind both IMA-638 and IL-13Rα2 simultaneously. In contrast to the IL-13Rα2–expressing A375 cells, no surface staining with IMA-638 was seen with HT-29 cells (2), which express IL-13Rα1 but not IL-13Rα2, suggesting that IL-13 interaction with IL-13Rα1 did not have sufficient stability to bridge IMA-638 to the surface of the cell.

FIGURE 4.

Dose-dependent binding of IMA-638 to IL-13Rα2 in the presence of IL-13. A375 cells were incubated overnight with 5, 20, or 50 ng/ml IL-13 (open histograms) or no added IL-13 (filled histograms). The next day, cells were stained with biotinylated control Ab (IVIG), IMA-638, or IMA-026, followed by PE-labeled streptavidin. Geometric mean fluorescence intensity is indicated in the IMA-638–staining panels.

FIGURE 4.

Dose-dependent binding of IMA-638 to IL-13Rα2 in the presence of IL-13. A375 cells were incubated overnight with 5, 20, or 50 ng/ml IL-13 (open histograms) or no added IL-13 (filled histograms). The next day, cells were stained with biotinylated control Ab (IVIG), IMA-638, or IMA-026, followed by PE-labeled streptavidin. Geometric mean fluorescence intensity is indicated in the IMA-638–staining panels.

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To reduce the potential for effector activity, both IMA-638 and IMA-026 contain mutations in the lower hinge of the H chain C region (2). The effectiveness of these mutations was assessed by comparing the Fc-mediated effector activity of IMA-638 with a wild-type Fc revertant. The wild-type Fc revertant had full binding to A375 cells in the presence of IL-13, as demonstrated by its ability to compete with biotinylated IMA-638 for cell surface binding (Fig. 5A). Fc-mediated effector activity was evaluated in Ab-dependent cellular cytotoxicity (ADCC) assays run using IL-13–loaded A375 cells as targets and NK-enriched PBMCs as effectors. The wild-type Fc revertant triggered cytolysis of A375 cells in a dose-dependent manner (Fig. 5B). This activity required IL-13 (Fig. 5C), consistent with binding of the Ab to IL-13 and not directly to the cells. In contrast, IMA-638 did not induce detectable ADCC, confirming that the Fc mutations eliminated the potential for effector activity. These experiments were repeated using HT-29 cells, which express IL-13Rα1 and not IL-13Rα2. There was no detectable cytolysis of this cell line either with IMA-638 or the wild-type Fc revertant (Fig. 5D), consistent with the observed lack of detectable IMA-638 binding to IL-13 captured by IL-13Rα1 (2). Because IMA-026 does not bind to A375 cells, even in the presence of IL-13 (Fig. 4), it would not be expected to mediate ADCC activity. This was confirmed using A375 cells (data not shown).

FIGURE 5.

IMA-638 lacks Fc-mediated ADCC activity. A, A375 human melanoma cells were preloaded with 3 ng/ml human IL-13. The indicated concentration of IMA-638 (○) or wild-type Fc revertant (▴) was bound to the cells in the presence of 3 μg/ml biotinylated IMA-638 for 30 min at 4°C. Cells were stained with PE-streptavidin, and binding of biotinylated IMA-638 was analyzed by flow cytometry. IVIG was run as a control human Ab of irrelevant specificity (■). B, ADCC in a 5-h [51Cr]-release assay using NK cell-enriched human PBMCs as effector cells and IL-13–loaded A375 cells as target cells, in the presence of IMA-638 (○) or wild-type Fc revertant (▴). C, Cytotoxicity of the wild-type Fc revertant against A375 cells in the presence or absence of IL-13. D, ADCC using NK cell-enriched human PBMCs as effector cells and IL-13–loaded HT-29 cells as targets in the presence of IMA-638 (○) or wild-type Fc revertant (▴).

FIGURE 5.

IMA-638 lacks Fc-mediated ADCC activity. A, A375 human melanoma cells were preloaded with 3 ng/ml human IL-13. The indicated concentration of IMA-638 (○) or wild-type Fc revertant (▴) was bound to the cells in the presence of 3 μg/ml biotinylated IMA-638 for 30 min at 4°C. Cells were stained with PE-streptavidin, and binding of biotinylated IMA-638 was analyzed by flow cytometry. IVIG was run as a control human Ab of irrelevant specificity (■). B, ADCC in a 5-h [51Cr]-release assay using NK cell-enriched human PBMCs as effector cells and IL-13–loaded A375 cells as target cells, in the presence of IMA-638 (○) or wild-type Fc revertant (▴). C, Cytotoxicity of the wild-type Fc revertant against A375 cells in the presence or absence of IL-13. D, ADCC using NK cell-enriched human PBMCs as effector cells and IL-13–loaded HT-29 cells as targets in the presence of IMA-638 (○) or wild-type Fc revertant (▴).

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Given that IL-13Rα2 functions to internalize bound IL-13, we asked whether the presence of IMA-638 or IMA-026 could affect the efficiency of cytokine internalization through this receptor. A375 cells were incubated with IL-13 for 24 h at 37°C, in the presence of IMA-638, IMA-026, or control human IgG. At the end of the incubation, the concentration of residual IL-13 was assayed in the culture medium, using assays that detect IL-13 in the presence or absence of each Ab (Fig. 6A, 6B). In the presence of control Ab, IL-13 was completely depleted within this time frame (Fig. 6C, 6D). In the presence of IMA-638, IL-13 could still be taken up into the cells, such that ∼50% of the starting concentration was depleted after 24 h (Fig. 6C, 6D). Increasing the concentration of IMA-638 did not increase the extent of IL-13 depletion beyond ∼50% (Fig. 6C, 6D). IMA-026 was more effective at blocking IL-13 uptake. At high concentrations of IMA-026, >90% of the input IL-13 remained in the extracellular medium (Fig. 6). Similar results were seen at relatively low (1 ng/ml; Fig. 6C) and high (10 ng/ml; Fig. 6D) concentrations of IL-13.

FIGURE 6.

Anti–IL-13 Abs differentially affect IL-13 internalization. Assays were developed to detect IL-13 in the presence or absence of anti-IL-13 Abs: IMA-638 (A) or IMA-026 (B). Addition of IMA-638 or IMA-026 at concentrations up to 10 μg/ml did not affect detection of IL-13. A375 cells were incubated overnight at 37°C with 1 ng/ml (C) or 10 ng/ml (D) human IL-13, in the presence of the indicated concentration of control human Ig, IMA-638, or IMA-026. The extracellular medium was collected and assayed for IL-13 using ELISAs developed to detect the cytokine in the presence of anti–IL-13 Ab.

FIGURE 6.

Anti–IL-13 Abs differentially affect IL-13 internalization. Assays were developed to detect IL-13 in the presence or absence of anti-IL-13 Abs: IMA-638 (A) or IMA-026 (B). Addition of IMA-638 or IMA-026 at concentrations up to 10 μg/ml did not affect detection of IL-13. A375 cells were incubated overnight at 37°C with 1 ng/ml (C) or 10 ng/ml (D) human IL-13, in the presence of the indicated concentration of control human Ig, IMA-638, or IMA-026. The extracellular medium was collected and assayed for IL-13 using ELISAs developed to detect the cytokine in the presence of anti–IL-13 Ab.

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Confocal microscopy was used to further explore IL-13 binding and internalization through IL-13Rα2. FLAG-tagged human IL-13 bound to the surface of IL-13Rα2–bearing A375 cells (Fig. 7A). As a negative control, we confirmed that FLAG-tagged human IL-4 did not bind to these cells (Fig. 7B). Consistent with findings from flow cytometry, IL-13 bound to the cell surface in the presence of IMA-638 (Fig. 7C) but not in the presence of IMA-026 (Fig. 7E). In contrast, IL-4 did not localize to the surface of A375 cells in the presence of either IMA-638 (Fig. 7D) or IMA-026 (Fig. 7F). Compared to staining in the absence of Ab (Fig. 7A), IMA-026 greatly reduced the amount of IL-13 bound to the cell surface (Fig. 7E), whereas IMA-638 did not (Fig. 7C). IMA-638 could also be visualized staining the cell surface (Fig. 7G), colocalized with IL-13 (Fig. 7C). This is consistent with the model that IMA-638 does not stain the cells directly but binds to IL-13 interacting with IL-13Rα2 on the cell surface. In contrast, IMA-026 did not stain the cell surface, even in the presence of IL-13 (Fig. 7H).

FIGURE 7.

Binding of IL-13 and anti–IL-13 Ab to A375 cells by confocal microscopy. Binding of FLAG-tagged human IL-13 (A, C, E) or human IL-4 (B, D, F) to A375 cells following overnight incubation at 37°C with the indicated cytokine. Cells were stained with Cy3-labeled anti-FLAG Ab (red), To-PRO 3 nuclear stain (blue), and biotinylated control human Ig (A, B), biotinylated IMA-638 (C, D), or biotinylated IMA-026 (E, F), followed by Alexa Fluor 488-streptavidin (green). A, B, D, and F show the overlay of red (cytokine), green (Ab), and blue (nuclear) staining. C and E show the overlay of red (cytokine) and blue (nuclear) staining. The overlay of red (cytokine), green (Ab), and blue (nuclear) staining for IMA-638 with IL-13 (G) and IMA-026 with IL-13 (H). Two images are shown per panel. Original magnification ×630 (occular ×10, objective ×63).

FIGURE 7.

Binding of IL-13 and anti–IL-13 Ab to A375 cells by confocal microscopy. Binding of FLAG-tagged human IL-13 (A, C, E) or human IL-4 (B, D, F) to A375 cells following overnight incubation at 37°C with the indicated cytokine. Cells were stained with Cy3-labeled anti-FLAG Ab (red), To-PRO 3 nuclear stain (blue), and biotinylated control human Ig (A, B), biotinylated IMA-638 (C, D), or biotinylated IMA-026 (E, F), followed by Alexa Fluor 488-streptavidin (green). A, B, D, and F show the overlay of red (cytokine), green (Ab), and blue (nuclear) staining. C and E show the overlay of red (cytokine) and blue (nuclear) staining. The overlay of red (cytokine), green (Ab), and blue (nuclear) staining for IMA-638 with IL-13 (G) and IMA-026 with IL-13 (H). Two images are shown per panel. Original magnification ×630 (occular ×10, objective ×63).

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The cells were permeabilized and stained for internalized IL-13, which was readily distinguished from cell surface cytokine, as shown in Fig. 8A and 8B. Control Ab had no effect on this internalization (Fig. 8C). IMA-026 prevented IL-13 internalization into IL-13Rα2–expressing A375 cells (Fig. 8D). In contrast, IMA-638 allowed IL-13 uptake, which was reduced compared with the no-Ab control (Fig. 8E), consistent with findings from IL-13–uptake assays (Fig. 6). IMA-638 itself was internalized along with the IL-13 and showed a similar staining pattern (Fig. 8F).

FIGURE 8.

IL-13 and Ab internalization by confocal microscopy. A, Staining for cell surface IL-13. A375 cells were incubated overnight at 37°C with FLAG-tagged IL-13 and then stained with Cy3 anti-FLAG at 4°C to label cytokine bound to the cell surface. B, Staining for intracellular IL-13. A375 cells were fixed and permeabilized, and the intracellular cytokine was stained with Cy3 anti-FLAG. To study effects of anti–IL-13 Abs on IL-13 internalization, A375 cells were incubated for 24 h with FLAG-tagged IL-13 along with biotinylated Abs: control human Ig (C), IMA-026 (D), IMA-638 (E, F). The cells were fixed, permeabilized, and stained with Cy3 anti-FLAG (red), Alexa Fluor 488-streptavidin (green), and nuclear stain To-PRO 3 (blue). C and D, Overlay of three-color staining. E, IL-13 staining with Cy3 anti-FLAG overlaid with To-PRO 3. F, Ab staining with streptavidin overlaid with To-PRO 3. Two images are shown per panel. Original magnification ×630 (occular ×10, objective ×63).

FIGURE 8.

IL-13 and Ab internalization by confocal microscopy. A, Staining for cell surface IL-13. A375 cells were incubated overnight at 37°C with FLAG-tagged IL-13 and then stained with Cy3 anti-FLAG at 4°C to label cytokine bound to the cell surface. B, Staining for intracellular IL-13. A375 cells were fixed and permeabilized, and the intracellular cytokine was stained with Cy3 anti-FLAG. To study effects of anti–IL-13 Abs on IL-13 internalization, A375 cells were incubated for 24 h with FLAG-tagged IL-13 along with biotinylated Abs: control human Ig (C), IMA-026 (D), IMA-638 (E, F). The cells were fixed, permeabilized, and stained with Cy3 anti-FLAG (red), Alexa Fluor 488-streptavidin (green), and nuclear stain To-PRO 3 (blue). C and D, Overlay of three-color staining. E, IL-13 staining with Cy3 anti-FLAG overlaid with To-PRO 3. F, Ab staining with streptavidin overlaid with To-PRO 3. Two images are shown per panel. Original magnification ×630 (occular ×10, objective ×63).

Close modal

Endosomal staining was visualized using organelle lights reagent (Fig. 9A). The staining pattern for internalized IL-13 was consistent with endosomal localization (Fig. 9B). Internalized IMA-638 was also distributed in a similar staining pattern (Fig. 9C). In contrast, staining for IL-13Rα2 showed a diffuse intracellular pattern (Fig. 9D), as previously described (3436).

FIGURE 9.

Intracellular staining patterns for IL-13 and anti–IL-13 are consistent with endosomal localization. A375 cells were fixed, permeabilized, and stained with Alexa Fluor 568-phalloidin to define the actin cytoskeleton, To-PRO 3 to stain the nucleus, as well as organelle lights endosomal stain (A), IL-13–FLAG followed by FITC–anti-FLAG (B), IL-13–FLAG and biotinylated IMA-638 followed by Alexa Fluor 488-streptavidin (C), or biotinylated anti–IL-13Rα2 followed by Alexa Fluor 488-streptavidin (D). Three images are shown per panel. Original magnification ×630 (occular ×10, objective ×63).

FIGURE 9.

Intracellular staining patterns for IL-13 and anti–IL-13 are consistent with endosomal localization. A375 cells were fixed, permeabilized, and stained with Alexa Fluor 568-phalloidin to define the actin cytoskeleton, To-PRO 3 to stain the nucleus, as well as organelle lights endosomal stain (A), IL-13–FLAG followed by FITC–anti-FLAG (B), IL-13–FLAG and biotinylated IMA-638 followed by Alexa Fluor 488-streptavidin (C), or biotinylated anti–IL-13Rα2 followed by Alexa Fluor 488-streptavidin (D). Three images are shown per panel. Original magnification ×630 (occular ×10, objective ×63).

Close modal

Healthy subjects and mild asthmatics were treated with anti-human IL-13 Abs IMA-638 or IMA-026 in separate dose-ranging and allergen-challenge studies. These Abs have very similar affinities and neutralization activity for IL-13 (2) and achieved comparable serum concentrations in these studies (3), in accordance with their similar pharmacokinetic characteristics (37). Both Abs were safe and well tolerated (3). Despite these similarities, assays that detect IL-13 in the presence or absence of these Abs revealed ∼10-fold higher concentrations of circulating IL-13 following dosing with IMA-026. No IL-13 biological activity was detectable, because the IL-13 was presumably bound to Ab. Because the major difference between these Abs is the epitope of IL-13 that they recognize, we investigated how epitope specificity could contribute to these differences in circulating IL-13 concentration. We propose that the inhibition of IL-13 interaction with cell surface IL-13Rα2 in the presence of IMA-026, but not IMA-638, could have led to the elevated IL-13 concentrations in subjects dosed with IMA-026.

Both Abs are potent neutralizers of IL-13 bioactivity through the IL-13Rα1/IL-4Rα complex, but IMA-026 inhibits IL-13 interaction with IL-13Rα2, whereas MA-638 allows this interaction to occur (2). We observed strong binding of IMA-638 to IL-13, which was captured by IL-13Rα2–expressing A375 cells. Because bridging through IL-13 could target IMA-638 to the surface of IL-13Rα2–expressing cells, we addressed whether the Ab could target Fc-mediated effector responses toward those cells. To minimize the potential for effector activity, the Fc regions of both IMA-638 and IMA-026 contain mutations in the lower hinge region of IgG1 Fc (3840). The effectiveness of these mutations was confirmed by demonstrating that IMA-638 lacked ADCC activity toward IL-13–loaded, IL-13Rα2–expressing A375 cells, whereas the same Ab with wild-type Fc targeted ADCC to these cells in the presence of IL-13. Neither form of the Ab mediated ADCC against HT-29 cells, which express IL-13Rα1 but not IL-13Rα2. The interaction of IL-13 with IL-13Rα1 is of much lower affinity than is the interaction with IL-13Rα2 (41); presumably, it is not sufficiently stable to result in effective labeling of cells with IMA-638.

IL-13 is rapidly internalized through cell surface IL-13Rα2 (28, 42). A model system using IL-13Rα2–expressing A375 human melanoma cells showed this IL-13 internalization mechanism effectively depleted extracellular IL-13 in a dose- and time-dependent manner. Initial concentrations of up to 18 ng/ml extracellular cytokine, which far exceed the sub-pg/ml levels found in healthy subjects and asthmatics (31), were reduced to undetectable titer after 48 h exposure to IL-13Rα2–expressing A375 cells. We found that IMA-026 completely antagonized this process at a high concentration. In contrast, even at a high concentration, IMA-638 did not block >50% of the IL-13 uptake. Confocal microscopy confirmed that IL-13 was efficiently internalized into IL-13Rα2–expressing A375 cells and that IMA-638 could be internalized along with the cytokine. Although the modulation of IL-13 uptake through IL-13Rα2 is a possible contributing factor, our results cannot be taken as conclusive evidence that this mechanism was responsible for the differences in IL-13 captured by IMA-638 and IMA-026 in human clinical studies. In particular, the quantitative results seen in vitro cannot be directly extrapolated to the in vivo situation. Nevertheless, our findings indicated that the amount of IL-13 uptake blocked by IMA-638 is saturable, such that increasing concentrations of Ab do not trap increasing concentrations of IL-13. In contrast, IMA-026 blocks IL-13 uptake in a dose-dependent manner that is not saturable. Given the pg/ml IL-13 concentrations and μg/ml Ab concentrations found in human subjects (3), differential effects on IL-13 internalization could help to account for the large differences in IL-13 titer seen in subjects treated with IMA-638 compared with IMA-026. These findings suggested that internalization through IL-13Rα2 functions to regulate IL-13 concentrations in humans.

Studies in mice support that IL-13Rα2 may represent a key component of the normal clearance mechanism for IL-13 generated in vivo. Arima et al. (29) showed that IL-13Rα2–bearing cells can deplete extracellular IL-13, detectable as a reduction in IL-13 bioactivity. However, because mice also have a soluble form of IL-13Rα2, the influence of cell surface IL-13Rα2 on IL-13 internalization may be difficult to discern. Humans lack soluble IL-13Rα2 (26, 43), and, as a result, the cell surface form may be a more critical regulator of IL-13 activity. The highly efficient uptake of IL-13 through IL-13Rα2 observed in our studies and in other human cell systems (28) suggested that IL-13Rα2 could be characterized both as a decoy and a scavenger receptor, a dual role that has been ascribed to a small number of other receptor systems. IL-1RII, a decoy for IL-1, mediates the rapid and efficient internalization of the cytokine by human neutrophils (44). D6, a nonsignaling receptor for CC chemokines on endothelial cells, also functions as a scavenger, and it mediates efficient internalization and degradation of chemokines (4547). The combined role of decoy and scavenger establishes a highly efficient system for limiting biological activity coupled with depletion.

In human clinical studies, administration of IMA-026 resulted in ∼10-fold higher titers of circulating IL-13 than was seen following treatment with IMA-638. Our findings with IL-13Rα2–expressing cells provide a mechanistic framework for interpretation of this observation. We showed that IMA-638 allows some degree of IL-13 internalization through IL-13Rα2, whereas IMA-026 does not. Thus, in the presence of IMA-638, the active IL-13Rα2 receptor-mediated IL-13–elimination pathway is only partially inhibited. In the presence of IMA-026, this pathway is completely inhibited, resulting in higher total concentrations of IL-13. The physiologically low concentration of IL-13, below 1 pg/ml (31), may be controlled by rapid IL-13 clearance via IL-13Rα2 rather than a slow well-controlled production of IL-13. These findings point to cell surface IL-13Rα2 as a potentially critical mediator of IL-13 clearance in humans.

We thank Dr. Joseph Balthasar (University of Buffalo) and Drs. Lynette Fouser and Kyri Dunussi-Joannopoulos (Pfizer) for helpful discussions.

Abbreviations used in this article:

ADCC

Ab-dependent cellular cytotoxicity

IVIG

intravenous immunoglobulin.

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M.K., K.M., T.C., and L.T. are current employees of Pfizer Research. The other authors have no financial conflicts of interest.