Journal of Immunological Methods 241 (2000) 43–59
`
`www.elsevier.nl/locate/jim
`
`Monoclonal antibodies against the human interleukin-11
`receptor alpha-chain (IL-11Ra) and their use in studies of
`human mononuclear cells
`
`a
`b
`c
`b
`Chrystel Blanc , Patricia Vusio , Karin Schleinkofer , Olivier Boisteau ,
`c
`b
`c
`c
`´
`¨
`¨
`Stefan Pflanz , Stephane Minvielle , Joachim Grotzinger , Gerhard Muller-Newen ,
`a ,*
`c
`b
`´
`Peter C. Heinrich , Yannick Jacques , Felix A. Montero-Julian
`aImmunotech: A Beckman–Coulter Company,130 av. de Lattre de Tassigny, BP 177,13276 Marseille Cedex 9, France
`bINSERM U463, Institut de Biologie,9 Quai Moncousu,44035 Nantes Cedex 01, France
`c
`¨
`Institut f ur Biochemie RWTH Aachen, Klinikum Pauwelstr.30,52057, Aachen, Germany
`
`Received 23 December 1999; received in revised form 15 March 2000; accepted 4 April 2000
`
`Abstract
`
`A panel of 14 hybridoma cell lines secreting monoclonal antibodies against the human interleukin-11 receptor alpha chain
`(hIL-11Ra) was obtained using two different approaches. Two antibodies were raised against peptides of the N- and
`C-terminal sequences, respectively, of the extracellular part of the hIL-11Ra. Another group of 12 antibodies was generated
`against a hybrid protein consisting of the extracellular part of the hIL-11Ra fused to mature full-length human IL-2. All
`these antibodies recognized native hIL-11Ra and most also recognized the denatured receptor on immunoblots after
`SDS–PAGE. Four different epitopes were identified on the extracellular part of the hIL-11Ra. One epitope, defined by the
`E27 antibody, is located at the N-terminus and the other three epitopes are clustered in the membrane-proximal, C-terminal
`region. The antibodies defining epitopes I and II recognized membrane-bound hIL-11Ra expressed in gp130/hIL-11Ra-co-
`transfected Ba/ F3 cells. The E27 antibody cross-reacted with murine IL-11Ra,
`in agreement with the fact
`that
`the
`N-terminal region is highly conserved between species. The other 13 antibodies all recognized a region between amino acids
`319 and 363, which is the membrane-proximal part of the hIL-11Ra. This region, which is less conserved between mouse
`and human, is shown here to be an immunodominant region. Anti-IL-11Ra monoclonal antibodies, which have not been
`described previously enabled us to explore the expression and tissue distribution of IL-11Ra on human peripheral blood
`mononuclear cells and cell lines. The antibodies provide powerful tools for the study of the regulation and function of the
`receptor.
`2000 Elsevier Science B.V. All rights reserved.
`
`Keywords: Interleukin-11, Interleukin-11 receptor; Cytokine receptors; Monoclonal antibody
`
`interleukin-11
`interleukin; IL-11Ra,
`Abbreviations: CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; IL,
`receptor alpha chain; mAb, monoclonal antibody; PBS, phosphate-buffered saline; SDS–PAGE, sodium dodecyl polyacrylamide gel
`electrophoresis
`*Corresponding author: Tel.: 133-4-9117-2752; fax: 133-4-9117-2753.
`E-mail address: montero@immunotech.fr (F.A. Montero-Julian)
`
`0022-1759/00/$ – see front matter
`PII: S0022-1759( 00 )00194-0
`
`2000 Elsevier Science B.V. All rights reserved.
`
`Lassen - Exhibit 1015, p. 1
`
`(cid:211)
`(cid:211)
`

`

`44
`
`C. Blanc et al. / Journal of Immunological Methods 241(2000)43–59
`
`1. Introduction
`
`Interleukin-11 (IL-11) was first described as a
`hematopoietic cytokine produced by bone marrow
`stromal cells (Paul et al., 1990). A variety of cells
`produce IL-11. It is involved in the development and
`proliferation of hematopoietic progenitor stem cells,
`as well as at different stages of hematopoiesis, such
`as the enhancement of megakaryocytopoiesis and
`platelet formation (Neben and Turner, 1993; Du and
`Williams, 1997). Its role in platelet formation recent-
`ly led to its therapeutic use in the prevention of
`thrombocytopenia
`associated with chemotherapy
`(Tepler et al., 1996). IL-11 exerts pleiotropic ac-
`tivities in different tissues, including the induction of
`acute phase proteins (Baumann and Schendel, 1991),
`the stimulation of the differentiation of B lympho-
`cytes into immunoglobulin-secreting cells (Anderson
`et al., 1992), the development of osteoclastic cells
`(Girasole et al., 1994), a role in neuronal develop-
`ment (Mehler et al., 1993), and the induction of
`protective effects in the intestinal mucosa (Booth and
`Potten, 1995). Recently, IL-11 was implicated in
`female reproduction (Robb et al., 1998) and was
`shown to have potent anti-inflammatory properties
`(Trepicchio et al., 1996; Hill et al., 1998).
`IL-11 has been grouped into the interleukin-6
`(IL-6) family (for reviews, see, Kishimoto et al.,
`1995; Heinrich et al., 1998). This family is com-
`posed of IL-6,
`leukemia inhibitory factor (LIF),
`oncostatin M (OSM), ciliary neurotrophic factor
`(CNTF) and cardiotrophin-1 (CT-1). All members of
`this
`cytokine
`family share
`the
`transmembrane
`glycoprotein gp130 as a common signal transducing
`subunit. An IL-11 receptor alpha-chain (IL-11Ra)
`binds IL-11 with low affinity (K |10 nmol/l) and is
`d
`responsible for ligand-binding specificity (Hilton et
`al., 1994). The IL-11 /IL-11Ra complex triggers the
`association of two gp130 molecules (Yin et al.,
`1993), leading to the formation of a high-affinity
`receptor (K |400–800 pmol/l) (Hilton et al., 1994)
`d
`able to transduce signals via the Janus kinase (Jak) /
`signal
`transducer and activator of
`transcription
`(STAT) signaling pathway (Dahmen et al., 1998).
`The IL-11Ra gene has been cloned in the mouse
`´
`(Hilton et al., 1994) and human (Cherel et al., 1995;
`Nandurkar et al., 1996). The mouse genome contains
`two distinct IL-11Ra loci (Bilinski et al., 1996); one
`
`of these genes is expressed strongly in the testis and
`is restricted to some mouse strains. In the human,
`´
`only one locus has been identified (Cherel et al.,
`1996; Van Leuven et al., 1996), but two different
`cDNAs were isolated, both coding for a membrane-
`anchored receptor with one lacking the cytoplasmic
`´
`portion (Cherel et al., 1995, 1996). The two isoforms
`are both active (Lebeau et al., 1997). The sequence
`homology between murine and human IL-11Ra is
`84% at the amino acid level. At present, there is little
`information about
`the expression of IL-11Ra re-
`ceptor protein, and the existence of a soluble form of
`the receptor remains to be demonstrated. A func-
`tional role for IL-11 and for its a-receptor moiety
`and possible involvement in human disease has not
`been established. In order to address these questions,
`highly specific reagents directed at the IL-11Ra have
`to be developed, and we report here the generation
`and characterization of a panel of monoclonal anti-
`bodies against human IL-11Ra. We took advantage
`of these antibodies to analyze IL-11Ra receptor
`surface expression on peripheral blood mononuclear
`cell subsets and numerous human cell lines.
`
`2. Materials and methods
`
`2.1. Reagents and antibodies
`
`Fine chemicals, unless otherwise stated, were from
`Merck (Darmstadt, Germany) and cell culture re-
`agents from Biowhittaker (Gagny, France). Human
`recombinant
`IL-2 and IL-11, biotinylated rabbit
`polyclonal anti-IL-11 and recombinant murine IL-
`11RaFc chimera were from R&D Systems (Abin-
`gdon, UK). Restriction enzymes were purchased
`from Eurogentec (Brussels, Belgium). HAT reagent
`was purchased from Sigma (St. Louis, MO). Rabbit
`polyclonal anti-murine IL-11R (N20) was purchased
`from Santa Cruz Biotechnology (California, USA).
`This antibody was generated using a peptide coding
`for the first 20 amino acids of the extracellular
`domain of the murine IL-11Ra and displayed 95%
`homology with the corresponding hIL-11Ra peptide.
`Peroxidase-, biotin- or phycoerythrin-conjugated
`goat anti-mouse and goat anti-rabbit antibodies, as
`well as streptavidin conjugated to phycoerythrin or
`peroxidase, were obtained from Immunotech (Marse-
`
`Lassen - Exhibit 1015, p. 2
`
`

`

`C. Blanc et al. / Journal of Immunological Methods 241(2000)43–59
`
`45
`
`ille, France). Anti-human Fc-domain antibody was
`from Jackson Laboratories (New Jersey, USA).
`
`lines were maintained in RPMI-1640 containing 10%
`(v/v) FCS supplemented with additives.
`
`2.2. Cells and cell culture
`
`The IL-3-dependent mouse pro-B-cell line Ba/ F3
`and the stable transfectant Ba/F3/130/ IL-11Ra
`expressing both the human signal
`transduction
`subunit gp130 and the human IL-11 receptor a were
`generated as described (Lebeau et al., 1997). Ba/ F3
`cells were maintained in RPMI-1640 containing 10%
`(v/v) heat-inactivated fetal calf serum (FCS), sup-
`plemented with additives (2 mM glutamine, 1 mM
`sodium pyruvate, 0.1 mM non-essential amino acids,
`500 U/ml penicillin/ streptomycin) and 2% (v/ v)
`WEHI-3-conditioned medium as a source of murine
`IL-3. Transfected cells were cultured in the same
`medium supplemented with 1 ng/ml of human
`recombinant IL-11 instead of IL-3. Mouse myeloma
`cell line X63.Ag.653 was maintained in Dulbecco’s
`modified Eagle’s medium (DMEM) supplemented
`with 10% FCS and additives. The hybridoma cell
`lines were grown in DMEM supplemented with 20%
`FCS, additives and HAT (hypoxanthine–amino-
`pterin–thymidine). Chinese hamster ovary (CHO)
`cells deficient in dehydroxyfolate reductase (DHFR
`2/ 2) were grown in DMEM/F12 medium with
`10% FCS, 2 mM glutamine, 500 U/ml penicillin /
`streptomycin. Transfected CHO cells were grown in
`RPMI-1640 supplemented with 10% FCS, additives
`and 750 mg/ml of G418. Meg01 and MO7E
`megakaryoblastic leukemia, HEL and TF1 erythro-
`leukemia, KG1a bone marrow acute myelogenous
`leukemia, K562 chronic myelogenous
`leukemia,
`HL60 promyelocytic leukemia, THP-1 monocytic
`leukemia, NK3.3 NK cells, RPMI8866 B-cell lym-
`phoma, Daudi Burkitt lymphoma, Peer and Molt13
`leukemic T-cell
`lymphoma, MG63 and Saos2 os-
`teosarcoma, SW620 colon adenocarcinoma, INT407
`embryonic intestine, A375 melanoma, MDA-MD-
`157 and MCF-7 breast carcinoma, JAR choriocar-
`cinoma, SVK14 transformed keratinocyte, HELA
`epithelial carcinoma, SRJH30 rhabdomyosarcoma,
`SK-N-SH, SK-N-MC and IMR32 neuroblastoma,
`SNB-19, A172 and U373MG glioblastoma cell lines
`were obtained from the ATCC (Rockville, MD). The
`WM35 Melanoma cell line was provided by Dr M.
`Herlyn (Wistar Institute, Philadelphia, PA). All cell
`
`2.3. Preparation of soluble IL-11R–IL-2 fusion
`protein, transfection of CHO cells and purification
`of sIL-11R–IL-2
`
`A cDNA encoding a soluble form of the human
`IL-11Ra (amino acids 1–363) was generated by
`polymerase chain reaction (PCR) amplification from
`´
`the complete cDNA of human IL-11Ra (Cherel et
`al., 1995) as template, with 59-GGAATTCGAAAT-
`GAGCAGCAGCTGCTCAG-39 as sense primer and
`59-TGCATGCATCACAGAGTCCCTGTGATCA-39
`as anti-sense primer. The fragment was cloned into a
`Bluescript plasmid coding for human Interleukin-2
`(hIL-2) opened with EcoRI/PstI. The resulting fused
`sIL-11R–IL-2 gene contains two additional codons
`as linker between the sIL-11Ra and IL-2 genes,
`coding for the dipeptide Met–Gly. The sequence of
`the hybrid sIL-11R–IL-2 gene was confirmed by
`sequencing. The sIL-11R–IL-2 was sub-cloned into
`the mammalian expression vector pKCR6 between
`the XhoI/NotI sites. The Bluescript plasmid con-
`taining hIL-2 and the vector pKCR6 were provided
`by Dr M. Bonneville (INSERM, Nantes).
`DHFR 2/ 2 CHO cells were co-transfected with
`both the pKCR6 sIL-11R–IL-2 plasmid and with the
`pCDNA3 plasmid (Invitrogen, Netherlands) which
`carries the neomycin resistance gene, thus providing
`an additional means of selection. The transfection
`was performed using LipofectAMINE PLUS Re-
`agent (Life Technologies) according to the manufac-
`turer’s protocol. Clones producing sIL-11R–IL-2
`protein were detected using a commercial enzyme
`immunoassay for the detection of human IL-2 (Im-
`munotech). One clone, 1.22, was selected for its
`maximal production of sIL-11R–IL-2 of about 300
`ng/ml, based on the measurement of the immuno-
`reactivity of human IL-2 in the supernatant. The
`supernatant of clone 1.22 was collected after 7 days,
`when the cell monolayer was confluent and was
`concentrated by precipitation with ammonium sulfate
`at 60% saturation. An IL-2-immunoaffinity column
`was prepared by grafting an anti-IL-2 mAb (IL-2.66,
`Immunotech) onto CNBr-activated Sepharose (Phar-
`macia, Uppsala, Sweden) following the protocol of
`the manufacturer. The concentrated supernatant was
`
`Lassen - Exhibit 1015, p. 3
`
`

`

`46
`
`C. Blanc et al. / Journal of Immunological Methods 241(2000)43–59
`
`dialyzed and loaded onto the column. The column
`was washed exhaustively with 50 mM Tris, 0.15 M
`NaCl pH 7.5 and eluted with 50 mM sodium acetate
`pH 4.2. The concentration of sIL-11R–IL-2 was
`determined in the fractions collected with the IL-2
`immunoassay. Active fractions were pooled, dialyzed
`against PBS and stored at 2208C. Purity of the
`product was assessed by SDS–PAGE (Miniprotean
`Ready Gels, BioRad, Ivry-sur-Seine, France). Pro-
`teins were stained with Coomassie Blue or trans-
`ferred onto a nitrocellulose membrane (Hybond
`ECL, Amersham, Les Ullis, France) using a transblot
`cell (BioRad). After incubation with blocking re-
`agent (Roche Molecular Biochemicals, Germany),
`the membrane was probed with either the anti-IL-2
`mAb described above or anti-murine IL-11Ra rabbit
`polyclonal
`(N20),
`followed by incubation with
`appropriate peroxidase-conjugated secondary antibo-
`dies. Development was performed using an ECL
`(Enhanced ChemoLuminescence) kit (Amersham).
`
`2.4. Preparation of the soluble IL-11R/FP
`
`The soluble IL-11R/FP was prepared as described
`(Pflanz et al., 1999). Briefly, this protein contains
`domains II and III of the hIL-11Ra (amino acids
`109–318) followed by a 21 amino acid spacer linked
`to mature human IL-11. This protein does not
`contain the Ig-like domain of the hIL-11Ra. The
`gene encoding this protein was cloned into pPICZaA
`plasmid (Invitrogen). Competent P. pastoris GS115
`were transfected with pPICZaAIL-11R/ FP applying
`the LiCl method according to the manufacturer’s
`instructions. Transfected cells were selected and
`conditions for the expression of IL-11R/FP were
`optimized.
`
`2.5. Preparation of a soluble domain III protein of
`hIL-11Ra (IL-11RD3), expression in E. coli,
`folding and purification.
`
`The domain III-encoding region (amino acids
`212–337) of the hIL-11Ra, IL-11RD3, was am-
`plified by PCR using hIL-11Ra-cDNA as a template
`´
`(Cherel et al., 1995). NcoI and BamHI sites were
`introduced into the 59- and 39-primers, respectively
`to make possible cloning of the amplified DNA in
`the corresponding sites of the E. coli expression
`
`vector pET8c/3d (Stratagene, La Jolla, CA, USA)
`(sense primer, 59-GGT GGT GCC ATG GAG AGC
`ATC TTG CGC CCT GAC-39; anti-sense primer
`59-CCG GAA GCT TAC TCC ACC TCT GGC TGC
`GT-39). The IL-11RD3 cDNA construct was con-
`firmed by restriction analysis and DNA sequencing.
`E. coli BL21 (DE3) (Studier et al., 1990) was
`transformed with the IL-11RD3 expression vector.
`Expression of the recombinant protein was induced
`by addition of isopropyl-b-D-thiogalactopyranoside
`(final concentration: 0.5 mM) for 3 h. Cells were
`harvested by centrifugation and resuspended in lysis
`buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA,
`0.2% Triton X-100, 1 mM dithiothreitol (DTT)).
`Complete lysis of bacteria was achieved by three
`freeze–thaw cycles followed by four steps of sonica-
`tion. The inclusion body pellet containing the recom-
`binant protein was purified through ten cycles of
`washing and centrifugation. Inclusion bodies were
`solubilized in 6 M guanidine–HCl, 50 mM Tris–
`HCl, pH 8.0, 100 mM DTT. The final concentration
`of the recombinant protein was approximately 5
`mg/ml. Solubilized inclusion bodies were submitted
`to an in vitro folding process by rapid 1/100 dilution
`in 20 mM Tris–HCl pH 8.5, 1 mM EDTA, 200 mM
`NaCl, 1 mM DTT. The folded recombinant protein
`was purified on a Superdex 75 (26/ 60) column
`(Amersham Pharmacia Biotech). Fractions were ana-
`lyzed by SDS–PAGE, pooled and concentrated.
`
`2.6. IL-11/sIL-11R–IL-2 binding assay
`
`Interaction of IL-11 with sIL-11R–IL-2 was mea-
`sured by an ELISA-based binding assay using the
`purified protein sIL-11R–IL-2 as described previous-
`ly (Tacken et al., 1999).
`
`2.7. Generation of hybridoma-producing
`monoclonal anti-hIL-11Ra antibodies
`
`2.7.1. Immunization of mice with peptides and
`screening
`Peptides (Genosys, Cambridge, UK) correspond-
`ing to amino acids 23–43 (SSPCPQAWGPPGVQYG-
`QPGRS) of the N-terminal region (peptide 1) and to
`amino
`acids
`345–363
`(PPRPSLQPHRRLLDH-
`RDSV) of the C-terminal region of the extracellular
`domain of the hIL-11Ra (peptide 2) were conjugated
`
`Lassen - Exhibit 1015, p. 4
`
`

`

`C. Blanc et al. / Journal of Immunological Methods 241(2000)43–59
`
`47
`
`to carrier proteins, ovalbumin or KLH (Keyhole
`Limpet hemocyanin) (Pierce, USA). BALB/c mice
`(Iffa Credo, Les Oncines, France) were immunized
`three times at 3-weekly intervals with 100 mg of
`either peptide conjugated to KLH, intraperitoneally
`in complete Freund’s adjuvant (CFA) (Sigma). Three
`weeks after the final
`injection, one of the mice
`received 50 mg of peptide conjugated to ovalbumin
`intravenously and was sacrificed 3 days later. Spleen
`cells were fused with mouse myeloma X63.Ag8.653
`using polyethylene glycol 1500 (Sigma) and selected
`in standard HAT medium. Hybridoma supernatants
`were screened on 96-well plates coated with peptides
`or with sIL-11R–IL-2 protein. Positive hybridomas
`were cloned under limiting dilution and two hybrid-
`omas were established: E27 directed against
`the
`N-terminal peptide and A39R against the C-terminal
`peptide. Both antibodies recognized not only the
`immunizing peptide but also the recombinant hybrid
`protein sIL-11R–IL-2. The isotypes were determined
`using a mouse isotyping kit (Amersham). Ascitic
`fluids were generated from both hybridomas and
`purified by affinity chromatography using protein L
`(Interchim, Montlucon, France) for E27 mAb which
`is of the IgA isotype or protein A (Pharmacia) for
`A39R mAb which is of the IgG1 isotype.
`
`2.7.2. Immunization of mice with sIL-11R–IL-2
`and screening
`BALB/c mice were immunized subcutaneously
`twice at 3-weekly intervals with 15 mg of sIL-11R–
`IL-2 in CFA. Three weeks after the second immuni-
`zation, one mouse received intravenously 5 mg of
`sIL-11R–IL-2 and was sacrificed 3 days later. Spleen
`cells were fused as described above. Hybridoma
`supernatants were screened both on 96-well plates
`coated with sIL-11R–IL-2 or hIL-2 and by cyto-
`metric
`analysis
`on Ba/F3/130/IL-11Ra cells.
`Twelve clones, E1.8, E12.7, E10.1, I12.3, C4.2, I7.4,
`D14.7, D16.1, E24.2, C8.7, B24.3, A3.4 were estab-
`lished. All mAbs were of the IgG1 isotype. Ascitic
`fluids were generated and mAbs purified using
`protein A Sepharose (Pharmacia).
`
`2.8. Epitope analysis of anti-IL-11Ra mAbs
`
`The definition of the epitopes recognized by the
`different anti-IL-11Ra mAbs was performed in an
`
`ELISA format by cross-pairing each mAbs with all
`other mAbs. Antibodies were biotinylated with
`biotin-N-hydroxy-succinimide-1 ester (Roche Diag-
`nostic)
`following the manufacturer’s instructions.
`The 96-well plates were coated with mAbs at 5
`mg/ml in PBS for 24 h at 48C and saturated with 3%
`BSA in PBS. Then 100 ml /well of CHO-derived
`sIL-11R–IL-2 supernatant at 30 ng/ml were incu-
`bated for 2 h at room temperature, followed by three
`washes with an automatic washer (SLT, Salzburg,
`Austria). One hundred ml/well of each biotinylated
`anti-IL-11Ra mAbs at 1 mg/ml (except A39R at 5
`mg/ml) were incubated for 2 h at room temperature.
`After three additional washes, 100 ml/well of per-
`oxidase-streptavidin (1/50,000) was added and the
`plates incubated for 1 h. The enzymatic activity was
`revealed using 100 ml/well of substrate (1% tetra-
`methybenzimide, 0.1% H O in 0.1 M sodium
`2
`2
`acetate, pH 5.5). After development in the dark for
`10 min, the reaction was stopped and the absorbance
`at 450 nm was measured using an ELISA reader
`(Molecular Device, UK).
`
`2.9. Surface plasmon resonance (SPR) studies
`
`The affinity and dissociation constants for each
`antibody were calculated by SPR studies with the
`BIAcore 2000 optical biosensor (BIAcore). Briefly,
`the chimeric sIL-11R–IL-2 protein was covalently
`linked to the activated carboxylated dextran matrix
`of the biosensor chip (CM5, BIAcore) via its primary
`amine groups, as recommended by the manufacturer.
`The coupling reaction was carried out for 7 min at a
`flow-rate of 5 ml/min at a sIL-11R–IL-2 concen-
`tration of 20 mg/ml and the chip was blocked with
`ethanolamine. A control chip was prepared by block-
`ing carboxymethyl groups directly with ethanol-
`amine. Concurrently, peptide 2 was covalently linked
`to another biosensor chip at a concentration of 2.3
`mg/ml. Anti-IL-11R mAbs were allowed to bind
`sequentially to sIL-11R–IL-2 or peptide 2 in Hepes-
`buffered saline. Concentration (a) of mAb bound to
`sIL-11R–IL-2 or peptide 2 and the reaction rates
`(da/dt) are given by the BIAlogue software. Six
`different mAb concentrations (1, 2, 5, 10, 20 and 50
`mg/ml) were run. Regeneration of the flow cells was
`achieved with 10 mM glycine HCl pH 1.8. Kinetic
`rate constants (k
`and k
`), as well as apparent
`
`on
`
`off
`
`Lassen - Exhibit 1015, p. 5
`
`

`

`48
`
`C. Blanc et al. / Journal of Immunological Methods 241(2000)43–59
`
`/k )
`(K 5 k
`equilibrium dissociation constants
`on
`d
`off
`were determined using BIAlogue kinetics evaluation
`software.
`
`streptavidin–peroxidase for 1 h. Development was
`performed by ECL.
`
`2.10. Flow cytometric analysis
`
`3. Results
`
`The parental Ba /F3 cell line and transfectant Ba /
`5
`F3/130/IL-11Ra (10 cells) were stained for 1 h at
`48C with either hybridoma supernatant or anti-IL-
`11R mAbs at concentrations ranging from 0.39 to
`100 mg/ml, washed twice in wash solution (PBS /
`0.2% BSA/0.02% NaN ) and incubated for 15 min
`3
`at 48C in the dark with phycoerythrin-conjugated
`goat-anti-mouse
`antibody
`(1 /200). Cells were
`washed twice more and fixed in PBS–0.5% parafor-
`5
`maldehyde. For cell lines, 10 cells were stained for
`1 h at 48C with 2 mg of E24.2 mAb, washed twice in
`wash solution and incubated with biotin-conjugated
`goat-anti-mouse antibody (1 /100). After 15 min,
`cells were washed twice more and incubated with
`phycoerythrin-conjugated streptavidin (1/100)
`for
`another 15 min. Cells were then washed and fixed.
`Peripheral blood mononuclear cells (PBMC) were
`purified from normal donors’ whole blood by Ficoll
`density gradient separation using a standard protocol.
`PBMC were stained using the same protocol as that
`employed for the cell
`lines at antibody concen-
`trations ranging from 1.6 to 100 mg/ml. In all
`experiments, non-specific monoclonal antibody bind-
`ing was controlled by using the IgG1 isotypic control
`(Immunotech)
`to set cursors. All samples were
`analyzed on a FACScan flow cytometer using Lysis
`II software (Becton Dickinson, Mountain View, CA).
`
`2.11. Immunoprecipitation of recombinant murine
`IL-11RaFc chimera
`
`The recombinant murine IL-11RaFc purchased
`from R&D was immunoprecipitated with anti-IL-
`11Ra mAbs and anti-human IgGFc mAb. To avoid
`cross-reactivity of the Fc region with protein A, we
`immunoprecipitated the complexes with protein L-
`Sepharose. The bound material was eluted by boiling
`and then analyzed by SDS–PAGE. Proteins were
`transferred onto nitrocellulose membranes as de-
`scribed above. Membranes were incubated with 1
`mg/ml of biotinylated anti-human IgGFc for 1 h,
`followed, after washing steps, by incubation with
`
`3.1. Characterization of the fusion protein sIL-
`11R–IL-2
`
`Cultures of transfected CHO cells that produced a
`soluble molecule, sIL-11R–IL-2, corresponding to a
`fusion of the extracellular domain of hIL-11Ra (Fig.
`1A) to hIL-2 were established (Fig. 1B). Analysis of
`the purified sIL-11R–IL-2 fusion protein revealed
`two bands at 59 and 62 kDa,
`respectively, by
`Coomassie Blue staining (Fig. 2A) and by immuno-
`blot with either an anti-IL-2 monoclonal antibody or
`a polyclonal anti-murine IL-11R antibody cross-
`reacting with hIL-11Ra, both under reducing and
`non-reducing conditions (Fig. 2B). Deglycosylation
`experiments showed that the two bands corresponded
`to two N-glycosylation states of the molecule (data
`not shown) in accord with the two predicted N-
`´
`glycosylation sites within human IL-11Ra (Cherel et
`al., 1995). Using plates coated with sIL-11R–IL-2
`and a non-blocking anti-hIL-11 biotinylated poly-
`clonal antibody,
`the fusion protein sIL-11R–IL-2
`was shown to retain its capacity to bind IL-11 with
`an ED50 (5 nM) comparable to the affinity already
`reported for IL-11 /IL-11Ra interaction (Hilton et
`al., 1994) (Fig. 3). No signal was detected when
`BSA was employed for coating instead of sIL-11R–
`IL-2 (data not shown).
`
`3.2. Epitope analysis of anti-IL-11Ra antibodies.
`
`Using either peptides corresponding to the N- and
`C-terminal regions of hIL-11Ra or the soluble fusion
`protein sIL-11R–IL-2, we raised monoclonal anti-
`bodies against the hIL-11Ra. The mapping analysis
`of the two anti-peptide antibodies, E27 and A39R, as
`well as of the 12 antibodies generated against sIL-
`11R–IL-2, was performed using a chequer board
`ELISA format. Table 1 summarizes the reactivity of
`the 14 anti-hIL-11Ra mAbs. Epitope I was recog-
`nized by E1.8, E12.7, E10.1, I12.3, C4.2, I7.4, D14.7
`and A39R antibodies, epitope II by D16.1, E24.2,
`C8.7 and B24.3 antibodies, and epitope III by a
`
`Lassen - Exhibit 1015, p. 6
`
`

`

`C. Blanc et al. / Journal of Immunological Methods 241(2000)43–59
`
`49
`
`Fig. 1. Schematic representation of primary and ternary structure of the recombinant proteins and peptides. (A) Full-length hIL-11Ra: Ser23
`to Leu422; domain I: Ser23 to Leu109; domain II: Gly110 to Gly223; domain III: Leu224 to Gly318; transmembrane domain: Glu364 to
`Trp390; and cytoplasmic domain: L391 to Leu422. The region between Thr319 and Val363 is predicted to be a connecting segment between
`the extracellular domain of the hIL-11Ra and the transmembrane domain. (B) sIL-11R–IL-2: Ser23 to Val363 of the hIL-11Ra. (C) Peptide
`1: Ser23 to Ser43, peptide 2: Pro345 to Val363 of the hIL-11Ra. (D) IL-11R/FP: Leu109 to Gly318 of the hIL-11Ra, followed by a 21
`amino acid linker and human IL-11 Pro29 to Leu199. (E). IL-11RD3: Leu212 to Glu337.
`
`single antibody, A3.4. Epitope IV was defined by
`antibody E27 which was produced against the N-
`terminal peptide (peptide1) (Fig. 1C). It was the only
`mAb that recognized this region. In the course of the
`mapping experiment,
`the A39R mAb against
`the
`C-terminal peptide was found to cluster with all the
`mAbs defining epitope I,
`therefore implying that
`epitope I overlaps with the C-terminal peptide (pep-
`tide 2) (Fig. 1C) and was confirmed by direct ELISA
`on peptide 2-coated plastic: anti-epitope I antibodies
`recognized the peptide, while anti-epitope II, III and
`IV antibodies did not (data not shown). Epitope I is
`therefore located close to the transmembrane part of
`the hIL-11Ra.
`In order to map epitopes II and III more precisely,
`we took advantage of two additional recombinant
`proteins: first, the IL-11R/FP protein described by
`Pflanz et al. (1999) which was previously reported to
`
`be functional (Fig. 1D); and secondly, a protein
`corresponding to an isolated soluble domain III of
`the hIL-11Ra,
`IL-11RD3 (Fig. 1E). As shown,
`neither IL-11R/ FP (Fig. 1D) nor IL-11RD3 (Fig.
`1E) protein contains the C-terminal region of peptide
`2. Analysis of the reactivities of the anti-IL-11Ra
`mAbs with these recombinant proteins was carried
`out by immunoblotting. In Fig. 4 are shown results
`for one representative antibody for each epitope. The
`sIL-11R–IL-2 protein which is recognized by all
`antibodies was used as the positive control (Fig. 4A).
`All antibodies defining epitopes II and III bound to
`IL-11RD3 (Fig. 4C), but not to IL-11R/FP (Fig.
`4B), suggesting that
`the antibodies recognize an
`epitope located between amino acids 319 and 337.
`As expected, the antibodies defining epitope I (C-
`terminal) recognized neither of the two proteins (Fig.
`4B and C).
`
`Lassen - Exhibit 1015, p. 7
`
`

`

`50
`
`C. Blanc et al. / Journal of Immunological Methods 241(2000)43–59
`
`Fig. 2. Characterization of the fusion protein sIL-11R–IL-2. (A) Purified sIL-11R–IL-2 was separated by SDS–PAGE under non-reducing
`(NR) and reducing (R) conditions, and stained with Coomassie blue. (B) Purified sIL-11R–IL-2 protein was separated by SDS–PAGE under
`non-reducing and reducing conditions, and transferred onto nitrocellulose. To detect the fusion protein, the membrane was probed with
`either monoclonal anti-IL-2 or polyclonal anti-murine IL-11Ra (N20) antibody. The complex was visualized with either peroxidase-
`conjugated goat anti-mouse or goat-anti-rabbit antibody, respectively, followed by ECL.
`
`3.3. Affinity measurements
`
`Using biosensor technology, the affinity constants
`of all antibodies for sIL-11R–IL-2 and the C-termi-
`nal peptide were determined (Table 2). Except A39R
`(K
`5677 nM), all antibodies displayed
`d sIL-11R – IL-2
`high affinity for sIL-11R–IL-2 with dissociation
`constants (K ) ranging from 1.84 to 24.7 nM. When
`d
`comparing the affinities of each epitope I antibody, it
`appeared that they were similar with two exceptions.
`One was mAb A39R which recognized peptide 2
`(K
`521.6 nM) better
`than sIL-11R–IL-2
`(K
`5677 nM). Conversely, mAb C4.2
`d sIL-11R – IL-2
`had
`higher
`affinity
`for
`the
`sIL-11R–IL-2
`a
`(K
`517.5 nM) than for the peptide 2
`d sIL-11R – IL-2
`(K
`5341 nM).
`
`d peptide 2
`
`d peptide 2
`
`Fig. 3. Binding of hIL-11 to sIL-11R–IL-2. The 96-well plates
`coated with sIL-11R–IL-2 were incubated with dilutions of hIL-
`11 for 2 h. A constant concentration (500 ng/ml) of biotinylated
`goat polyclonal anti-IL-11 antibody was added for 1 h, followed
`by streptavidin–peroxidase and substrate.
`
`3.4. Flow cytometric analysis on Ba/F3130/IL-
`11R tranfectants
`
`We carried out flow cytometric analysis on Ba/ F3
`cells transfected with the signal transducer gp130
`
`Lassen - Exhibit 1015, p. 8
`
`

`

`C. Blanc et al. / Journal of Immunological Methods 241(2000)43–59
`
`51
`
`Table 1
`Epitope analysis of anti-hIL-11Ra antibodies
`
`a
`
`a Clustering of antibodies directed against the hIL-11Ra was carried out using a chequer board in an ELISA format. Results are expressed
`as the absorbance read at 450 nm of each well.
`
`Fig. 4. Recognition of three different sIL-11Ra molecules by anti-hIL-11Ra mAbs representative of each epitope. Purified human
`sIL-11R–IL-2 (A), partially purified IL-11R/FP (B), and purified IL-11RD3 (C) proteins were separated by SDS–PAGE under non-reducing
`conditions (left
`lanes) and reducing conditions (right
`lanes), and transferred onto nitrocellulose. The membranes were probed with
`biotinylated anti-IL-11Ra mAbs as indicated or control antibodies at 1 mg/ml. Complexes were visualized with either streptavidin–
`peroxidase (1/50 000) or peroxidase-conjugated goat anti-rabbit antibody (1/2000) followed by ECL. The positive control antibody for
`IL11R/FP was polyclonal anti-IL-11, for sIL-11R–IL-2, the polyclonal anti-mouse IL-11Ra (N20). No positive control was available for
`IL-11R/D3 but we used N20 as a negative control.
`
`Lassen - Exhibit 1015, p. 9
`
`

`

`52
`
`C. Blanc et al. / Journal of Immunological Methods 241(2000)43–59
`
`Table 2
`Affinity constants of anti-hIL-11Ra mAbs measured by BIAcore
`
`a
`
`Epitope
`
`Antibody
`
`K (nM) for
`d
`sIL-11R–IL-2
`
`K (nM) for
`d
`peptide 2 C-terminal
`
`4.25
`24.5
`34
`6.57
`341
`32.2
`16
`21.6
`
`I
`
`II
`
`III
`
`IV
`
`E1.8
`E12.7
`E10.1
`I12.3
`C4.2
`I7.4
`D14.7
`A39R
`
`B24.3
`D16.1
`E24.2
`C8.7
`
`A3.4
`
`E27
`
`1.84
`12.8
`22.8
`1.96
`17.5
`21.2
`17.5
`677
`
`7.06
`7.41
`7.65
`8.26
`
`24.7
`
`22.5
`
`a
`
`The sIL-11R–IL-2 protein and peptide 2 were immobilized on
`the sensor chip and the mAbs were analysed in the BIAcore
`system. Dissociation constants (K ) of mAbs was determined
`d
`using the kinetic constant evaluation BIAlogue.
`
`and the membrane-bound hIL-11Ra. These cells
`express hIL-11Ra at the cell surface and proliferate
`in response to hIL-11 as already reported (Lebeau et
`al., 1997). The flow cytometric titration curves are
`shown in Fig. 5. mAb A39R did not stain the
`Ba/F3-transfected cell line at all, while E27 gave
`very weak staining, which might be due to its isotype
`
`(data not shown). The antibodies defining
`(IgA)
`epitope II (E24.2, B24.3, D16.1 and C8.7 mAbs)
`stained membrane-bound hIL-11Ra most strongly.
`The mean fluorescence intensity was higher and
`obtained at lower concentrations than with epitope I
`and III antibodies (Fig. 5). It may be noted that
`antibody C4.2 behaved differently from the other
`epitope I mAbs, with a titration curve closer to those
`of the epitope II mAbs.
`
`3.5. Recognition of murine IL-11Ra by anti-human
`IL-11Ra antibodies
`
`A chimeric protein consisting of the extracellular
`domain of the murine IL-11Ra (1–345) and the Fc
`region of a human IgG was used in immuno-
`precipitation experiments to check the cross-reactivi-
`ty of the anti-IL-11Ra antibodies with murine IL-
`11Ra. All mAbs were precipitated by protein L and
`further incubated with the chimeric protein. The
`complexes were separated by SDS–PAGE and de-
`tected by immunoblotting with a biotinylated anti-
`human