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Molecular Immunology 44 (2007) 916–925

A human monoclonal IgG1 potently neutralizing thepro-inflammatory cytokine GM-CSF

Eva-Maria Krinner 1, Tobias Raum 1, Silke Petsch, Sandra Bruckmaier, Ioana Schuster,Laetitia Petersen, Ronny Cierpka, Derege Abebe, Michael Mølhøj, Andreas Wolf,

Poul Sørensen 2, Mathias Locher, Patrick A. Baeuerle ∗, Julia HeppMicromet AG, Staffelseestr. 2, D 81477 Munich, Germany

Received 1 February 2006; received in revised form 23 March 2006; accepted 27 March 2006Available online 11 May 2006

bstract

The pro-inflammatory cytokine GM-CSF is aberrantly produced in many autoimmune and chronic inflammatory human diseases. GM-CSFeutralization by antibodies has been shown to have a profound therapeutic effect in animal models of rheumatoid arthritis, inflammatory lungiseases, psoriasis and multiple sclerosis. Moreover, the absence of GM-CSF in null mutant mice ameliorates or prevents certain of these diseases.ere we describe the biophysical and biological properties of a human anti-GM-CSF IgG1 antibody designated MT203, which was derived byhage display guided selection. MT203 bound with picomolar affinity to an epitope on human and macaque GM-CSF involved in high-affinityeceptor interaction. As a consequence, the antibody potently prevented both GM-CSF-induced proliferation of TF-1 cells with a sub-nanomolarC50 value and the production of the chemokine IL-8 by U937 cells. MT203 neutralized equally well recombinant (r) human (h) GM-CSF fromscherichia coli and yeast, and also normally glycosylated GM-CSF secreted by human lung epithelial cells in response to IL-1� stimulation.urthermore, MT203 significantly reduced both survival and activation of peripheral human eosinophils as may be required for effective treatment

f inflammatory lung diseases. The antibody did not show a detectable loss of neutralizing activity after 5 days in human serum at 37 ◦C. Basedn its favorable properties, MT203 has been selected for development as a novel anti-inflammatory human monoclonal antibody with therapeuticotential in a multitude of human autoimmune and inflammatory diseases.

2006 Elsevier Ltd. All rights reserved.

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eywords: Monoclonal antibody; GM-CSF; Cytokine; Eosinophil; Inflammati

. Introduction

Few biological anti-inflammatory treatments are availableoday that specifically target the innate arm of the immune sys-em, while most therapies in clinical use and under developmentarget cells of the adaptive immune response, i.e., T and B cells.M-CSF has now been recognized as a key activator of innate

mmunity and, as such, is involved in chronic stages of inflam-

atory and autoimmune diseases where macrophages, granulo-

ytes, neutrophils, eosinophils and dendritic cells can contributeo tissue damage and disease progression (Hamilton, 2002).

∗ Corresponding author. Tel.: +49 89 895277 601; fax: +49 89 895277 205.E-mail address: patrick.baeuerle@micromet.de (P.A. Baeuerle).

1 Equally contributing first authors.2 Present address: LEO Pharma A/S, Industriparken 55, DK-2750 Ballerup,enmark.

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eatment

GM-CSF is a 23 kDa glycoprotein with a four alpha helicalundle structure (Diederichs et al., 1991) that binds to a het-rodimeric receptor composed of subunits belonging to the typecytokine receptor family (Hercus et al., 1994). GM-CSF was

riginally described as a potent stimulator of growth and differ-ntiation of granulocyte and macrophage precursor cells in vitroSheridan and Metcalf, 1973). Subsequent studies revealed thatt also stimulates proliferation and activation of mature immuneells as well as of antigen-presenting dendritic cells to increaseheir functional capacity in combating infections. GM-CSF isberrantly overproduced in a multitude of pro-inflammatory andutoimmune human diseases, and addition of recombinant GM-SF was found to aggravate such diseases (de Vries et al., 1991;

iehn et al., 1992; Kotsimbos et al., 1997; Openshaw et al., 2000;ousa et al., 1993).

Numerous animal models have shown that neutralization ofM-CSF by murine GM-CSF-specific antibodies has significant

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herapeutic potential. In mice immunized to develop collagen-nduced arthritis (CIA), anti-GM-CSF mAb treatment not onlymeliorated arthritis when treatment was started prior to dis-ase onset of clinical disease, but the arthritis score was reducedn mice with established disease (Cook et al., 2001). GM-CSFnockout mice were found to be resistant to development ofIA and experimental autoimmune encephalomyelitis (EAE),widely used model for human multiple sclerosis (Campbell

t al., 1998; McQualter et al., 2001). In addition, anti-GM-CSFntibody is both a potent preventive and curative therapeutic forAE in mice. In several mouse models of lung inflammation,nti-GM-CSF antibody treatment reduced acute and chronicnflammatory symptoms. Likewise, treatment with a GM-CSF-eutralizing antibody of flaky skin (fsn/fsn) mice, a model fortudying hyperproliferative inflammatory alterations of the skinquivalent to psoriasis in human, resulted in significant reduc-ion of inflammation and acanthosis (Schon et al., 2000).

Although monoclonal hybridoma antibodies against humanM-CSF have been generated in the past, no humanized or fullyuman form of such an antibody has yet been described thatould meet the high standards that industry, regulatory authori-

ies and patients are demanding for novel antibody therapeutics.pecifications for a therapeutic anti-human GM-CSF mono-lonal antibody include high affinity and neutralizing potential,ow off-rate, low immunogenicity, high stability and cross-eactivity with primate GM-CSF. Here we describe for the firstime a human, high-affinity IgG1 that potently neutralizes sev-ral biological activities of human GM-CSF. This antibody,alled MT203, showed robust binding to human GM-CSF irre-pective of the glycosylation status of the cytokine. Furthermore,

T203 is of high stability, cross-reacts with macaque GM-CSFnd appeared to fulfill critical specifications as requested forhe development of a therapeutic GM-CSF neutralizing anti-ody. In humans, possible disease indications for treatmentith MT203 include rheumatoid arthritis (RA), asthma andther forms of lung inflammation, multiple sclerosis (MS) andsoriasis.

. Materials and methods

.1. Generation of MT203

A human single-chain antibody (scFv) was generated byhage display and guided selection as described elsewhereKrinner et al., submitted for publication). Briefly, a human lighthain variable repertoire was combined with the VH domain ofparental rat GM-CSF neutralizing antibody. By phage dis-

lay selection, a GM-CSF neutralizing rat/human scFv wasdentified. In a second step, a VH domain pairing with theelected human light chain was selected from a human VH reper-oire that preserved the complementary determining region 3 ofhe parental rat VH. Phage display selection led to the iden-ification of a potently neutralizing human scFv. VH and VL

omains of this scFv were used to construct a human IgG1,alled MT203, by recombinant fusion with Ckappa, CH1, CH2nd CH3 domains essentially as described before (Raum et al.,001).

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unology 44 (2007) 916–925 917

.2. Expression and purification of MT203

For initial characterization, MT203 was produced in tran-iently transfected human HEK293-F cells. Vectors encodinghe heavy and light chain of MT203 were co-transfected inEK293-F cells using the 293 fectin reagent (Invitrogen,arlsruhe, Germany) according to the manufacturer’s instruc-

ions. In brief, DNA and 293 fectin were separately dilutedn OptiMEM Medium (Gibco, Karlsruhe, Germany), then

ixed and incubated for 30 min at room temperature. TheNA/293-fectin mix was then added to 293F cells adjusted

o 1 × 106 cells/mL in Freestyle 293 Expression MediumInvitrogen, Karlsruhe, Germany). Cells were gently shaken fordays at 37 ◦C and 8% CO2. Culture supernatant was harvestedy spinning down the cells. MT203 was purified from theulture supernatant by protein A chromatography according totandard protocols for the isolation of immunoglobulins.

.3. Surface plasmon resonance analysis

Association and dissociation rate constants were determinedy surface plasmon resonance on a BIAcoreTM 2000 instru-ent (Biacore AB, Freiburg, Germany) using CM5 sensor chips.fter activation of the sensor chip with NHS/EDC, immobi-

ization of rhGM-CSF (LeukineTM) on the chip was done bynjection of 10 �g/mL rhGM-CSF in 0.01 M sodium-acetate,H 4.7. Response units binding experiments were performed bynjecting serially diluted samples at a flow rate of 5 �L/min andBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA,.005% surfactant P20) as running buffer at 25 ◦C. Dissociationas monitored for 100 s. The association and dissociation rates

ka [1/Ms] and kd [1/s], respectively, were calculated using theIAevaluation software (3.2 RC1) with a 1:1 Langmuir bind-

ng equation. The equilibrium dissociation constant KD [M] wasalculated from kd/ka.

.4. ELISA

rhGM-CSF (LeukineTM), rhG-CSF or rhM-CSF at 1 �g/mLas immobilized on microtiter plates (Maxisorb, Nunc, Wies-aden, Germany). After blocking the wells with PBST contain-ng 3% BSA, MT203 was added in a dilution series and incu-ated for 1 h at room temperature. Detection of bound MT203as accomplished by a goat-anti-huIgG antibody conjugated

o horseradish peroxidase (Dianova, Hamburg, Germany). TheLISA was developed with ABTS substrate (Roche, Mannheim,ermany) and absorbance was measured at 405 nm.

.5. Proliferation inhibition assay

Propagation of TF-1 cells (DSMZ, Braunschweig, Germany;CC 334) was done in RPMI 1640, 10% fetal calf serum (FCS),

n the presence of 2.5 ng/mL rhGM-CSF. Determination of the

roliferation-inducing potential of GM-CSF was done by addi-ion of GM-CSF in a dilution series ranging from 100 ng/mLo 0.5 pg/mL without MT203. For the proliferation inhibitionssay, TF-1 cells were seeded at a density of 0.9 × 104 cells/well

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n RPMI 1640 (containing 10% FCS, but lacking rhGM-CSF) in96-well flat bottom microtest plate after washing twice with 1×BS. A final concentration of 0.3 ng/mL rhGM-CSF was used

o stimulate TF-1 cell proliferation. Alternatively, conditionedulture supernatant from IL-1� treated BEAS-2B was used fortimulation, resulting in a final concentration of 0.2 ng/mL hGM-SF. MT203 was added to inhibit proliferation in a dilution

eries ranging from 10 �g/mL to 50 ng/mL. After incubation for2 h at 37 ◦C in 5% CO2, the proliferative status was determinedy adding WST-1 reagent (Roche, Mannheim, Germany). Viableells were quantitated by measuring the absorbance at 450 nm.ata were analyzed and fitted for half-maximal inhibition of pro-

iferation (IC50) using the software package GraphPadPrism4.

.6. IL-8 inhibition assay

U-937 cells (DSMZ, Braunschweig, Germany; ACC 5) pro-uce IL-8 upon stimulation with GM-CSF. RPMI1640/10% FCSas used for cultivation. 1.8 × 105 cells were seeded in a 96-wellicrotest plate per well. To examine IL-8 production depending

n the GM-CSF concentration, GM-CSF was added in a dilutioneries ranging from 100 ng/mL to 0.001 ng/mL.

The inhibiting effect of MT203 on IL-8 production of U937ells was monitored during stimulation with a final concentra-ion of 1 ng/mL GM-CSF. MT203 was added in a dilution seriesesulting in final concentrations of 10 �g/mL to 10 pg/mL. Afterncubation for 16 h at 37 ◦C and 5% CO2, cells were spun downy centrifugation. Culture supernatants were harvested and ana-yzed to determine the concentration of IL-8 using the OptEIAuman IL-8 Elisa set (BD Biosciences Heidelberg, Germany).LISA detection was carried out according to the manufacturer’s

nstructions. For later quantitation of the IL-8 concentration aerial dilution of the IL-8 standard provided by the manufac-urer was performed in parallel. IL-8 concentration in the cultureupernatant samples was calculated according to the standardurve. Data were fitted for half-maximal inhibition of IL-8 pro-uction by a non-linear regression curve fit using the softwareackage GraphPad Prism4.

.7. Serum stability

The MT203 antibody was diluted in 50% human serum atconcentration of 50 �g/mL and incubated at 37 ◦C for up to

15 h. Samples were taken at different time points and storedt −20 ◦C. For analysis samples were thawed at the same timend tested for their biological activity in the TF-1 proliferationnhibition assay as described above.

.8. Cultivation of BEAS-2B cells and induction ofM-CSF production

BEAS-2B cells were propagated in BEBM-medium sub-tituted with the BEGM Bullet Kit (Cambrex, Verviers, Bel-

ium) but cultured in RPMI 1640, 10% FCS in the presencef 50 ng/mL IL-1� (Strathmann Biotech, Hamburg, Germany)or induction of GM-CSF production. After 48 h incubation at7 ◦C, 5% CO2 the culture supernatant was analyzed for its GM-

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munology 44 (2007) 916–925

SF content using the OptEIA Human GM-CSF Elisa Set (BDiosciences, Heidelberg, Germany) according to the manufac-

urer’s instructions.

.9. BEAS-2B/TF-1 cell co-culture system

For co-culture experiments with TF-1 cells BEAS-2B cellsere seeded in RPMI 1640, 10% FCS at a density of 1 × 104 cellser well in a 96-well flat bottom plate. After adherence of theells overnight at 37 ◦C, 5% CO2, 1 × 104 TF-1 cells were addeder well and GM-CSF production was induced by 1 ng/mL IL-�. MT203 or an isotype control antibody was added to a finaloncentration of 5 �g/mL for inhibition of TF-1 proliferation.fter incubation for 72 h at 37 ◦C in 5% CO2, viable cells wereuantified by adding WST-1 reagent (Roche, Mannheim, Ger-any). The resulting colorimetric reaction indicating live cellsas measured by the absorbance at 450 nm. The absorption value

esulting from BEAS-2B cells cultured alone was determined inhe same experiment. This value was subtracted from the absorp-ion value determined from co-culture in order to determine thealue resulting from TF-1 cell proliferation.

.10. Isolation of eosinophils from peripheral blood

Eosinophils were isolated from peripheral blood from healthyonors. Freshly drawn, heparinized blood was mixed with.7 vol. of 6% Dextran T70 (Amersham, Freiburg, Germany) in.9% NaCl and incubated for erythrocyte sedimentation for 1 ht room temperature. The leukocyte-enriched fraction was lay-red onto Ficoll (Biochrom, Berlin, Germany) and centrifugedt 400 × g for 30 min. The mononuclear cell layer and Ficollolution was removed. Erythrocytes that were still present inhe granulocyte pellet were lysed by resuspending the pellet inater and restoring isotonicity by adding 0.35 vol. of 3.5% NaCl

fter 30 s. Granulocytes were pelleted and washed once in 1×BS. Anti-human CD16 microbeads (50 �L per 5 × 107 cells)Miltenyi, Heidelberg, Germany) were added to deplete CD16-ositive neutrophils from the granulocyte suspension and incu-ated for 30 min at 4 ◦C. The cell suspension was layered onto aACS cell separation column in a magnetic field. Flow-through

nd wash containing CD16-negative cells were collected, cen-rifuged at 300 × g for 10 min and resuspended in RPMI 1640,0% FCS. FACS analysis revealed that approximately 80% ofhe cells were CD16-negative, eosinophilic granulocytes. Con-aminating cells were identified as CD16- and CD3-positive cellsdata not shown).

.11. Eosinophil survival and survival-inhibition assay

For the eosinophil survival as well as for the survival-nhibition assay freshly isolated peripheral blood eosinophilsere seeded at a density of 5 × 104 cells/well in RPMI 1640/10%CS and Pen/Strep in a 96-well flat bottom microtest plate.

M-CSF was added in a dilution series ranging from 33 ng/mL

o 10 pg/mL to monitor the concentration-dependent eosinophilurvival. To analyze the inhibiting potential of MT203 on GM-SF-dependent eosinophil survival the antibody was added

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n a dilution series ranging from 10 �g/mL to 0.1 ng/mL. Anal concentration of 0.1 ng/mL GM-CSF was used to effectosinophil survival. After incubation for 72 h at 37 ◦C, 5% CO2

ST-1 reagent was added. The resulting colorimetric reac-ion corresponding to the portion of viable cells was quanti-ed by measuring the absorbance at 450 nm. The data werenalyzed and fitted for half-maximal inhibition of survivalIC50) using the non-linear regression curve fit of the prismoftware.

.12. Analysis of CD69 expression on eosinophils

Eosinophils isolated from peripheral blood were cultivatedn 48-well plates at a density of 5× 105 cells/mL in RPMI 1640,0% FCS and Pen/Strep at 37 ◦C and 5% CO2. Incubation wasone without addition of cytokine or in presence of 0.1 ng/mLhGM-CSF (LeukineTM). 10 �g/mL MT203 were used for neu-ralization of GM-CSF. After incubation for 20 h or 3 days,espectively, cells were analyzed for CD69 surface expressiony flow cytometry.

For quantitation of MT203-mediated inhibition of CD69xpression eosinophils from peripheral blood were seeded atdensity of 5 × 104 cells/well in RPMI 1640, 10% FCS and

en/Strep in a 96-well flat bottom microtest plate. GM-CSFas added to a final concentration of 0.1 ng/mL to stimulate the

ells and a dilution series of MT203 was added ranging from0 �g/mL to 0.1 ng/mL. After incubation for 20 h at 37 ◦C, 5%O2 CD69 expression was determined by FACS analysis.

.13. Flow cytometry

Expression of CD69 on eosinophils was determined on aACS Calibur instrument (Becton Dickinson, Heidelberg, Ger-any). 1 × 105 cells were incubated with 5 �L of a FITC-

onjugated anti-human CD16 (clone 3G8, BD Biosciences,eidelberg, Germany) and a PE-conjugated anti-human CD69

ntibody (clone FN50, BD Biosciences, Heidelberg, Germany)ach for 1 h at 4 ◦C. As a negative control irrelevant, isotype-atched FITC- and PE-conjugated antibodies were used. After

ncubation, cells were washed twice with PBS, 1% FCS,.05% NaN3 and resuspended in 250 �L PBS, 1% FCS, 0.05%aN3. Propidium iodide was added to label dead cells infinal concentration of 1 �g/mL immediately before FACS

nalysis. Data interpretation was done using the CellQuestProoftware (BD Biosciences, Heidelberg, Germany). Propidiumodide-positive cells were excluded from analysis of CD69xpression.

. Results

.1. MT203 is specific for GM-CSF

We used ELISA to test whether MT203 can bind colony-

timulating factors other than GM-CSF. The three humanolony-stimulating factors G-CSF, M-CSF und GM-CSF weremmobilized on a microtiter plate and binding by MT203as detected through an enzyme-conjugated anti-human IgG

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ested in a dilution series by ELISA. Error bars give standard deviations fromuplicate experiments.

econdary antibody. A strong binding signal of MT203 wasbtained for GM-CSF, but no binding of the antibody to the twother colony-stimulating factors was detected (Fig. 1). Likewise,-CSF and M-CSF did not detectably compete with MT203inding to GM-CSF (data not shown).

.2. MT203 neutralizes human GM-CSF from differentources and macaque GM-CSF with equal potency

The neutralizing potential of MT203 was investigated by itsnhibitory effect on GM-CSF-dependent proliferation of humanF-1 cells. Human GM-CSF variants produced in E. coli, yeastnd human cells were used to investigate whether MT203 bind-ng to human GM-CSF was affected by their different gly-osylation. Macaque rGM-CSF produced in human cells wasested for cross-reactivity with MT203 for potential use of thisrimate species in pre-clinical toxicology studies. Naturally gly-osylated human GM-CSF was obtained by stimulation of theuman bronchial epithelial cell line BEAS-2B with 50 ng/mLL-1�. As determined by ELISA, the concentration of hGM-SF in BEAS-2B cell culture supernatant was approximately.2 ng/mL.

A dilution series of conditioned medium containing naturalGM-CSF, as well as recombinant hGM-CSF from E. coli andeast, and macaque GM-CSF were first tested for their potencyo induce TF-1 proliferation. All three variants of human GM-SF and macaque GM-CSF had very similar EC50 values ofF-1 stimulation. These were 10 pg/mL for E. coli, 15 pg/mL foreast and 36 pg/mL for human lung cell-produced hGM-CSF,nd 11 pg/mL for macaque GM-CSF, respectively (Fig. 2A).

The neutralizing activity of MT203 was then determined inhe presence of 0.3 ng/mL recombinant hGM-CSF, or 0.2 ng/mLhysiological hGM-CSF. After 72 h, the proliferative status of

F-1 cells in the presence of different MT203 concentrationsas quantified by a colorimetric reaction (Fig. 2B). MT203

nhibited GM-CSF-dependent proliferation of TF-1 cells at sub-anomolar concentrations apparently independent from the gly-

920 E.-M. Krinner et al. / Molecular Immunology 44 (2007) 916–925

Fig. 2. The effect of MT203 on GM-CSF-induced TF-1 cell proliferation.(A) GM-CSF-dependent proliferation of TF-1 cells. hGM-CSF from differentsources as well as macaque GM-CSF was tested for its ability to stimulate TF-1cell proliferation. After incubation for 72 h, viable cells were quantified by addi-tion of WST-1 reagent and measuring the absorbance at 450 nm. (B) Analysis ofMT203 for neutralization of GM-CSF-induced TF-1 cell proliferation. TF-1 cellpMa

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Table 1Characteristics of MT203

Isotype Human IgG1Specificity Human and macaque GM-CSFAssociation rate constant, ka 3.6 ± 2.4 × 105 M−1 s−1

Dissociation rate constant, kd 1.7 ± 0.5 × 10−5 s−1

Equilibrium dissociation constant, KD 46 ± 34 pMSerum stability at 37 ◦C >5 days

IC50 (nM) 95% confidenceinterval (nM)

Inhibition of TF-1 proliferationrhGM-CSF 0.15 0.10–0.20Macaque GM-CSF 0.10 0.07–0.13

Inhibition of IL-8 production 0.20 0.10–0.30Inhibition of eosinophil survival 0.13 0.09–0.17Inhibition of eosinophil activation 0.22 0.08–0.60

Fig. 3. The effect of MT203 on GM-CSF-induced IL-8 production in U937cells. (A) rhGM-CSF-induced IL-8 production by U937 cells. Following incu-bation with rhGM-CSF for 16 h, IL-8 concentration in the culture supernatantwas determined by ELISA. (B) Dose-dependent inhibition of IL-8 production

roliferation was induced by 0.3 ng/mL GM-CSF (0.2 ng/mL for hGM-CSF).T203 was added in a dilution series. Viable cells were quantitated by WST-1

fter 72 h.

osylation status of the human cytokine. Half-maximal inhibi-ion (IC50) of TF-1 cell proliferation was observed at 0.15 nM

T203. MT203 showed an IC50 value of 0.1 nM with macaqueM-CSF, which has 95.3% sequence identity with human GM-SF (Fig. 2A; Table 1) (95% confidence interval: rhGM-CSF.10–0.20 nM; macGM-CSF 0.07–0.13 nM; Table 1).

The GM-CSF-neutralizing potential of MT203 was assessedn a second assay system by investigating the production of IL-8y the human monocyte cell line U937 in response to rhGM-SF. Fig. 3A shows the rhGM-CSF dose-dependent release of

L-8 into U937 culture supernatant after cytokine stimulationor 16 h. For quantification of the GM-CSF neutralizing effect of

T203, U937 cells were stimulated for 16 h with 1 ng/mL GM-

SF in the presence of varying concentrations of MT203. Theuman monoclonal antibody showed a potent inhibition of GM-SF-induced IL-8 production at a half-maximal concentration of.2 nM MT203 (95% confidence interval: 0.1–0.3 nM; Table 1),

by MT203. U937cells were stimulated with 1 ng/mL rhGM-CSF for 16 h inpresence of varying MT203 concentrations. The concentration of IL-8 in theculture supernatant was determined by ELISA. Error bars give standard devia-tions from duplicate experiments. Half-maximal neutralization was reached ata concentration of 0.2 nM MT203.

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hich was very similar to the concentration required for half-aximal inhibition of TF-1 cell proliferation.

.3. High-affinity GM-CSF binding by MT203

The association (ka) and dissociation rate constants (kd) forinding of MT203 to immobilized rhGM-CSF were determinedsing surface plasmon resonance spectroscopy.

Binding kinetics were determined with concentrations ofT203 ranging from 10 �g/mL to 1 pg/mL of purified pro-

ein. The independent fitting of the raw data resulted in dis-ociation and association rate constants that were used toalculate the equilibrium dissociation constant (KD). MT203ound to rhGM-CSF with an association rate constant ofa = 3.6 ± 2.4 × 105 M−1 s−1, a dissociation rate constant ofd = 1.7 ± 0.5 × 10−5 s−1, and an equilibrium binding constantf KD = 46 ± 34 pM (Table 1).

.4. Serum stability of MT203

Resistance to serum proteases is essential for long-term appli-ation of an antibody therapeutic. Here, we tested the biologi-al activity of MT203 following incubation of the antibody inuman serum at 37 ◦C for up to 5 days. Serum incubation had noetectable impact on the neutralizing activity of MT203 (Fig. 4).T203 retained for each incubation period its ability to inhibit

he GM-CSF-dependent proliferation of TF-1 cell with a half-aximal inhibition concentration of approximately 0.15 nM.

.5. MT203 neutralizes the GM-CSF-dependentroliferation of TF-1 cells in co-culture withL-1β-stimulated human bronchial epithelial cells

We investigated the neutralizing effect of MT203 in a cell co-ulture experiment, which is thought to reflect a paracrine cellctivation by GM-CSF. The GM-CSF-dependent cell line TF-1as cultured together with the adherent cell line BEAS-2B, a

ig. 4. Serum stability of MT203. MT203 was incubated in 50% human serumor 0 h, 24 h, 48 h and 115 h at 37 ◦C. Subsequently its neutralizing effect onhGM-CSF-dependent TF-1 cell proliferation was tested. TF-1 cell prolifera-ion was induced by 0.1 ng/mL rhGM-CSF. Viable cells were quantitated by

ST-1 reagent after 72 h. Error bars give standard deviations from duplicatexperiments.

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r presence of 1 ng/mL IL-l�. MT203 or an isotype control antibody (huIgG1)ere added at a final concentration of 5 �g/mL. After 72 h, the proliferative

tatus of cells was measured by addition of WST-1 reagent.

ronchial epithelial cell line that secretes GM-CSF upon stim-lation with IL-1�. Stimulation with 1 ng/mL IL-1� resultedn a GM-CSF concentration of 70 pg/mL in the BEAS-2B cul-ure supernatant after 24 h, and reached 130 pg/mL cytokinefter 72 h. Without stimulation, only low amounts of GM-CSFere detected after 72 h (ca. 25 pg/mL). Cultivation of TF-1

ells on a monolayer of adherent BEAS-2B cells resulted in aetectable increase of TF-1 proliferation cells over base line,hich was doubled by addition of 1 ng/mL IL-l� (Fig. 5). In theresence of 5 �g/mL MT203, TF-1 cell proliferation rate waseduced below the rate seen in the unstimulated co-culture whileddition of an isotype-matched control antibody had no suchffect.

.6. MT203 inhibits GM-CSF-dependent survival ofosinophils

One of the various biological activities of GM-CSF is pro-ongation of eosinophilic and neutrophilic granulocyte survivalEsnault and Malter, 2002; Lampinen et al., 2004). Because lungnflammatory diseases are associated with local accumulation ofosinophils, which play a substantial role in maintaining inflam-ation (Lantero et al., 1997), we tested efficacy of MT203 in

nhibiting GM-CSF-mediated eosinophil survival. Eosinophilsere isolated from peripheral blood of healthy donors by deple-

ion of CD16+ neutrophils from the granulocyte populationbtained by density gradient centrifugation and lysis of ery-hrocytes. Eosinophils were incubated with a dilution series ofhGM-CSF for up to 3 days to investigate a dependency of cellurvival on the cytokine. A half-maximal effective dose (EC50)f 0.02 ng/mL rhGM-CSF was determined (Fig. 6A). MT203

as added in dilution series to eosinophils cultured in presencef 0.1 ng/mL GM-CSF. A potent neutralizing effect of MT203as seen with a half-maximal inhibition of eosinophil survival at

n antibody concentration of 0.13 nM (95% confidence interval:.09–0.17 nM; Table 1).

922 E.-M. Krinner et al. / Molecular Im

Fig. 6. The effect of MT203 on eosinophil survival. (A) GM-CSF concentration-dependent survival of eosinophils. Eosinophils isolated from human peripheralblood were incubated with different amounts of rhGM-CSF for 3 days. Sur-vival of cells was measured by quantification of live cells using WST-1. Ahalf-maximal effective dose (EC50) of 0.02 ng/mL rhGM-CSF was calculatedby fitting the data. (B) MT203 dose-dependent inhibition of eosinophil survivalinduced through 0.1 ng/mL rhGM-CSF. Cells were incubated for 3 days and thenanalyzed for live cells using WST-1. The half-maximal inhibitory dose (IC50)ot

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f MT203 neutralizing 0.1 ng/mL rhGM-CSF derived from sigmoidal fitting ofhe data is 0.1 nM.

.7. MT203 inhibits GM-CSF-dependent activation ofosinophils

Besides eosinophil survival, we also investigated the effectf MT203 on GM-CSF-induced activation of eosinophils.D69 expression was found to be up-regulated on peripheralosinophils (CD16−) isolated from human blood followingtimulation for 20 h or 3 days with 0.1 ng/mL GM-CSF or.1 ng/mL GM-CSF, IL-3 plus IL-5, but not with 0.1 ng/mL IL-3lus IL-5 alone (Fig. 7A). Eosinophils cultured in the presencef medium alone showed now up-regulation of CD69. At bothime points, MT203 (10 �g/mL) almost completely preventedM-CSF-dependent activation of eosinophils, as seen by lackf CD69 expression in flow cytometry.

MT203 also reduced the percentage of live and activatedosinophils as monitored by propidium iodide staining ofD16−/CD69+ cells in the presence of 0.1 ng/mL GM-CSF.T203 reduced the percentage of activated cells from 35% to

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munology 44 (2007) 916–925

% after 1 day and from 43% to 3% after 3 days of cultiva-ion. In the presence of 0.1 ng/mL GM-CSF, IL-3 plus IL-5,he percentage of alive and activated eosinophils was reducedrom 32% to 8% and from 48% to 11% after 1 day and 3ays, respectively. Even though the up-regulation of CD69 wasompletely inhibited by MT203, higher numbers of restingosinophils (CD16−/CD69−) survived for 3 days in the presencef 0.1 ng/mL GM-CSF, IL-3 plus IL-5 compared to cells incu-ated with medium or GM-CSF alone (Fig. 7A, last column).he same was observed for cells incubated in the presence of.1 ng/mL IL-3 plus IL-5.

In dose finding experiments, MT203 was added in dilutioneries to eosinophils cultured in presence of 0.1 ng/mL GM-CSFFig. 7B). An inhibitory effect of MT203 on CD69-dependentedian fluorescence intensity (MFI) was observed at a half-aximal concentration of 0.22 nM MT203 (95% confidence

nterval: rhGM-CSF 0.08–0.6 nM, Table 1).

. Discussion

In this study, we describe for the first time a human mono-lonal antibody, MT203, neutralizing human and macaque GM-SF. MT203 showed ideal binding properties for neutralizing arotein ligand. The antibody specifically bound human GM-CSFith a dissociation constant of 46 pM, translating into biolog-

cal activities with half-maximal inhibitory constants between00 pM and 200 pM. The high affinity of MT203 was mainlyue to a very slow off-rate, which may assure a very stable andherefore long-lived antibody–ligand complex. The high affin-ty and slow off-rate of MT203 for GM-CSF are comparable tohat of adalimumab (Humira®) for TNF-�. Like MT203, adali-

umab, which is highly successful in treatment of rheumatoidrthritis (Schwartzman et al., 2004), is a human IgG1 antibody.M-CSF levels in diseased tissue are of similarly low level as

hose of TNF-�, suggesting that with comparable binding kinet-cs for their protein ligands and using the same IgG1 subtype,osing of MT203 could be similar as for the anti-TNF-� anti-ody.

GM-CSF exhibits a variety of glycosylation isoforms corre-ating with a range of biological activities (Cebon et al., 1990). Itas therefore deemed important to neutralize the cytokine inde-endently from its variable glycosylation. We found that bindingf MT203 to human GM-CSF was not affected by glycosyla-ion of the cytokine. Non-glycosylated rhGM-CSF produced in. coli was neutralized equally well by MT203 as was high-annose type glycosylated rhGM-CSF produced in yeast and

omplex type glycosylated hGM-CSF produced by human cells.We chose to develop a GM-CSF neutralizing antibody in

he human IgG1 format. This format has proven to be highlyffective in the neutralization of small protein ligands as exem-lified with several marketed antibodies neutralizing TNF-

(Remicade®, Enbrel®, Humira®) and vascular endothelialrowth factor (Avastin®). Binding to and internalization of IgG1

y Fc� receptors on local antigen-presenting cells or by theeticular endothelial system may be a means for rapid clear-nce of antibody–ligand complex. This route of clearance maye particularly important in inflamed target tissue, which is char-

E.-M. Krinner et al. / Molecular Immunology 44 (2007) 916–925 923

Fig. 7. The effect of MT203 on activation of human peripheral eosinophils by GM-CSF. Eosinophils were isolated from peripheral blood and incubated for 1 dayand 3 days in medium containing 0.1 ng/mL GM-CSF alone, a mix of 0.1 ng/mL GM-CSF, IL-3 plus IL-5, or 0.1 ng/mL IL-3 plus IL-5 only. MT203 was addedat a concentration of 10 �g/mL and the effect on GM-CSF induced CD69 expression and survival of eosinophils analyzed. (A) FACS dot plots showing doublestaining of cells with CD16-FITC/CD69-PE antibodies. Dead cells were excluded by staining with propidium iodide. Numbers indicate the percentage of liveCD69+ eosinophils corresponding to the upper left quadrant. (B) Dose-dependent inhibition of eosinophil activation by MT203. CD69 expression on eosinophilswas induced by 0.1 ng/mL GM-CSF. Median fluorescence intensity (FI) of CD69 staining was measured and plotted against the concentration of MT203. Thehalf-maximal inhibitory dose (IC50) of MT203 in the experiment shown, as derived from sigmoidal fitting of the data, was 0.2 nM.

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24 E.-M. Krinner et al. / Molecul

cterized by increased numbers of macrophages, dendritic cellsnd other cells bearing Fc� receptors. C kappa, CH1, CH2 andH3 domains of IgG1 are human and identical for all anti-odies of this isotype. Potential immunogenicity can thus onlyeside in variable domains of MT203. Disregarding comple-entary determining region (CDR3), VH and VL domains ofT203 showed 91% and 88% closeness to human germline,

espectively. Low immunogenicity of biologicals is consideredarticularly important for treatment of inflammatory diseases,hich present with an enhanced state of immune reactivity.GM-CSF exhibits a large variety of biological activities

ffecting most cell types of the innate immune system in eitherheir activity and/or survival (Bozinovski et al., 2002; Gajewskat al., 2003; Lampinen et al., 2004; Santiago-Schwarz et al.,001; Xu et al., 2003). For testing the GM-CSF-neutralizingctivity of MT203 we thus had to focus in this study on exem-lary activities of the cytokine covering its proliferative, genenductory, activating and survival functions by using variousell culture models. We found that MT203 prevented TF-1ell proliferation, IL-8 production by U937 cells and peripheralosinophil activation and survival as mediated by GM-CSF. Inll assays, MT203 showed half-maximal inhibition of GM-CSFunctions at sub-nanomolar concentration. A model system withigh physiological relevance may be the co-culture of adherentronchial epithelial cells with TF-1 reporter cells. GM-CSF isroduced therein at basal and Il-1�-enhanced levels, which isreventing apoptosis and stimulating proliferation of the co-ultured TF-1 cells in a paracrine fashion. MT203 was alsoctive in this setting, suggesting that the antibody can effec-ively and rapidly capture GM-CSF upon its immediate releasey epithelial cells and prior to receptor binding on TF-1 cellsithin a narrow intercellular space. With peripheral eosinophils

rom human donors, we studied MT203 activity with primaryells that play a key role in lung inflammatory diseases (Humblest al., 2004; Kankaanranta et al., 2000; Lampinen et al., 2004;oolley et al., 1994). These cells are dependent on GM-CSFith respect to both activation and survival. MT203 reduced the

urvival of peripheral eosinophils after 1 day from 35% to 8%,nd after 3 days from 43% to 3%. In addition, complete inhi-ition of activation of eosinophils from peripheral blood wasbserved upon addition of MT203. The fact that neutralizingnti-mouse GM-CSF antibodies were found highly effective innumber of lung inflammatory diseases including asthma mod-ls (Bozinovski et al., 2002, 2004; Cates et al., 2003, 2004;amashita et al., 2002) suggests that MT203 may have similarctivity by neutralizing human GM-CSF. Hence, in all systemsnalyzed, MT203 could potently neutralize various biologicalctivities of GM-CSF.

GM-CSF has just recently experienced a profound validations an anti-inflammatory disease target in a multitude of mouseodels (Bozinovski et al., 2002, 2004; Cates et al., 2003, 2004;ook et al., 2001; McQualter et al., 2001; Schon et al., 2000;amashita et al., 2002). With MT203, a human IgG1 antibody

otently neutralizing human GM-CSF is now available to studyhe impact of GM-CSF neutralization in primates and man. Thentibody fulfills all specifications required for a human antibodyt this early stage of development: high potency and stability,

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munology 44 (2007) 916–925

owest possible immunogenicity and cross-reactivity with pri-ate species.

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