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`effector function of an Fe region containing protein are known in the art and/or
`described herein.
`
`In one example, the Fe region is an IgG4 Fe region (1.e., from an IgG4 constant
`
`region), e.g., a human [gG4 Fe region. Sequences ofsuitable [gG4 Fe regions will be
`apparent to the skilled person and/or available in publically available databases(e.g.,
`available from National Center for Biotechnology Information).
`
`In one example, the constant region is a stabilized IgG4 constant region. The
`term “stabilized IgG4 constant region” will be understood to mean an IgG4 constant
`region that has been modified to reduce Fab arm exchangeor the propensity to undergo
`
`Fab arm exchange or formation of a half-antibody or a propensity to form a half
`antibody. “Fab arm exchange”refers to a type of protein modification for human IgG4,
`in which an IgG4 heavy chain and attached light chain (hal f-moleculc) is swapped for a
`
`heavy-light chain pair from another IgG4 molecule. Thus, IgG4 molecules may acquire
`
`two distinct Fab arms recognizing two distinct antigens (resulting in bispecific
`molecules). Fab arm cxchange occurs naturally iz vive and can be induced in vitro by
`purified blood cells or reducing agents such as reduced glutathione. A “half antibody”
`
`forms when an IgG4 antibody dissociates to form two molecules each containing a
`single heavy chain and a single light chain.
`In one example, a stabilized IgG4 constant region comprises a proline at
`
`position 241 of the hinge region according to the system of Kabat (Kabat e7 al.,
`Sequences of Protcins of Immunological
`Interest Washington DC United States
`Department of Health and Human Services, 1987 and/or 1991).
`This position
`corresponds to position 228 of the hinge region according to the EU numbering system
`(Kabat et al., Scquences of Protcins of Immunological Intcrest Washington DC Unitcd
`States Department of Health and Human Services, 2001 and Edelmanet al., Proc. Natl.
`Acad. USA, 63, 78-85, 1969).
`In human IgG4,
`this residue is generally a serine.
`Following substitution of the serine for proline, the IgG4 hinge region comprises a
`sequence CPPC.
`In this regard, the skilled person will be aware that the “hinge region”
`is a proline-rich portion of an antibody heavy chain constant region that links the Fe
`and Fab regions that confers mobility on the two Fab arms of an antibody. The hinge
`region includes cysteine residues which are involved in inter-heavy chain disulfide
`bonds.
`It is generally defined as stretching from Glu226 to Pro243 of human IgG1
`according to the numbering system of Kabat. Hinge regions of other IgG isotypes may
`be aligned with the IgG1 sequence by placing the first and last cysteine residues
`
`forming inter-heavy chain disulphide (S-S) bonds in the same positions (see for
`example WO2010/080538).
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`Additional examples of stabilized IgG4 antibodies are antibodies in which
`arginine at position 409 in a heavy chain constant region of human IgG4 (according to
`the EU numbering system)is substituted with lysine, threonine, methionine, or leucine
`
`(e.g., as described in WO2006/033386). The Fc region of the constant region may
`additionally or alternatively comprise a residue selected from the group consisting of:
`alanine, valine, glycine, isoleucine and Icucine at the position corresponding to 405
`
`(according to the EU numbering system). Optionally, the hinge region comprises a
`proline at position 241 (i.e., a CPPC sequence) (as described above).
`In another example, the Fe region is a region modified to have reduced effector
`
`10
`
`function, Le., a “non-immunostimulatory Fe region”. For example, the Fc region is an
`IgG1 Fe region comprising a substitution at one or more positions selected from the
`group consisting of 268, 309, 330 and 331.
`In another cxample, the Fe region is an
`
`IgGl Fe region comprising one or more of the following changes E233P, L234V,
`
`L235A and deletion of G236 and/or one or more of the following changes A327G,
`A3308 and P331S (Armouret al, Eur J Immunol. 29:2613-2624, 1999; Shiclds et al., J
`
`15
`
`Biol Chem. 276(9):6591-604, 2001). Additional examples of non-immunostimulatory
`
`I'c regions are described, for example, in Dall'Acquaef al., J dmmunol. 177 : 1129-1138
`2006; and/or Hezarch J Virol ;75: 12161-12168, 2001).
`
`In another example, the Fe region is a chimeric Fe region, e.g., comprising at
`
`least one Cy2 domain from an IgG4 antibody and at least one Cy3 domain from an
`TgG1 antibody, wherein the Fe region comprises a substitution al onc or more amino
`acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307,
`309, 323, 399. 409 and 427 (EU numbering) (e.g., as described in WO2010/085682).
`Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P,
`309K, 309M, 309P, 323F, 3998S, and 427F.
`
`Enhancing Effector Function
`In one example, an 1L-11Ra-binding protein of the present disclosure may
`induce effector function or enhanced effector function.
`
`In the context of the present disclosure, “effector functions” refer to those
`biological activities mediated by cells or proteins that bind to the Fe region (a native
`sequence Fe region or amino acid sequence variant Fe region) of an antibody that result
`in killing of a cell. Examples of effector functions induced by antibodies include:
`complement dependent cytotoxicity; antibody-dependent-ccll-mediated cytotoxicity
`(ADCC); antibody-dependent-cell-phagocytosis (ADCP); and B-cell activation.
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`In one example, an IL-11Ra-binding protein of the present disclosure binds to
`IL-11Ro on the surface of a cell in such a manner that it is capable of inducing an
`effector function, such as, ADCC or CDC.
`For example, the [L-11Ra-binding protein remains bound to the IL-11Ra on the
`surface of the cell for a time sufficient to induce an effector function, such as ADCC
`and/or CDC.
`
`In one example, an IL-11Ra-binding protein of the present disclosure is capable
`of inducing enhanced effector function, e.g., by virtue of a modified Fe region or by
`virtue of comprising a region capable of binding to an immune effector cell. For
`example, the level of effector function is increased compared to the level induced by a
`human IgG1 or IgG3 Fe region. Enhancing effector function induced by a IL-11Ra-
`binding protein of the disclosure may result in enhanced therapeutic or prophylactic
`effects, e.g., by not only blocking the action of IL-11Re but also by killing or depleting
`cclls causing a condition, c.g., by killing auto-reactive T cclls.
`In one example, the Fe region of an IL-11Ra-binding protein of the disclosure is
`modified to increase the level of effector function it is capable of inducing compared to
`the Fe region without the modification. Such modifications can be at the amino acid
`level and/or the secondary structural level and/or the tertiary structural level and/or to
`the glycosylation of the Fc region.
`The skilled addressee will appreciate that greater effector function may be
`manifested in any of a numberof ways, for example as a greater level of effect, a more
`sustained effect or a faster rate of effect.
`
`In one example, the Fc region comprises one or more amino acid modifications
`that increase its ability to induce enhanced effector function.
`In one example, the Fe
`region binds with greater affinity to one or more FcyRs, such as FcyRIIT.
`In one
`example,
`the Fe region comprise at least one amino acid substitution at a position
`selected from the group consisting of: 230, 233, 234, 235, 239, 240, 243, 264, 266, 272,
`274, 275, 276, 278, 302, 318, 324, 325, 326, 328, 330, 332. and 335, numbered
`according to the EU index of Kabat. In one example, the Fc region comprises the
`following aminoacid substitutions $239D/I332E, numbered according to the EU index
`of Kabat. This Fc region has about 14 fold increase in affinity for FeyRIJa compared.
`to a wild-type Fe region and about 3.3 increased ability to induce ADCC compared to a
`wild-type Kc region. In one example, the Kc region comprises the following amino acid
`substitutions $239D/A330L/I332E, numbered according to the EU index of Kabat.
`This Fe region has about 138 fold increase in affinity for FeyRII]a compared to a wild-
`type Fe region and about 323 fold increased ability to induce ADCC compared to a
`wild-type Fc region.
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`Additional aminoacid substitutions that increase ability of a Fe region to induce
`effector function are known in the art and/or described, for example, in US6737056 or
`US7317091.
`
`In one example, the glycosylation of the Fe region is altered to increase its
`ability to induce enhanced effector function.
`In this regard, native antibodies produced
`by mammaliancells typically comprise a branched, biantennary oligosaccharide that is
`generally attached by an N-linkage to Asn297 of the Cy2 domain of the Fc region. The
`oligosaccharide may include various
`carbohydrates,
`e.g., mannose, N-acetyl
`glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a
`GlcNAcin the “stem” ofthe biantennary oligosaccharide structure. In some examples,
`Fe regions according to the present disclosure comprise a carbohydrate structure that
`lacks fucose attached (directly or indirectly) to an Fe region,
`i.e., the Fe region is
`“afucosylated”. Such variants may have an improved ability to induce ADCC. Mcthods
`for producing afucosylated antibodics include, cxpressing the antibody or antigen
`binding fragment thereof in a cell line incapable of expressing a-1,6-fucosyltransferase
`(FUTS8)(e.g., as described in Yumane-Ohnuki et al., Biotechnol. Bioengineer. 87: 614-
`622, 2004), expressing the antibody or antigen binding fragment
`thereof in cells
`expressing a small interfering RNA against PUTS (e.g., as described in Mori ef al,
`Biotechnol. Bioengineer., 88: 901-908, 2004), expressing the antibody or antigen
`binding fragment
`thereof in cells incapable of expressing guanosine diphosphate
`(GDP)-mannose 4,6-dehydratase (GMD)
`(e.g., as described in Kanda et al.,
`J.
`Biotechnol., 130: 300-310, 2007). The present disclosure also contemplates the use of
`proteins having a reduced level of fucosylation, e.g., produced using a cell
`line
`modified to express B—(1,4)-N-acetylglucosaminyltransferase TI] (GnT-IID (e.g., as
`described in Umana et al., Nat. Biotechnol. 17: 176-180, 1999).
`Other methods include the use of cell lines which inherently produce antibodies
`capable of inducing enhanced Fe-mediated effector function (e.g. duck embryonic
`derived stem cells for the production of viral vaccines, WO2008/129058; Recombinant
`protein production in avian EBX® cells, WO 2008/142124).
`IL-l1Ro-binding proteins of the present disclosure also include those with
`bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the
`Fe region is bisected by GlcNAc. Such proteins may have reduced fucosylation and/or
`improved ADCC function. Examples of
`such proteins are described, e.g.,
`in
`US6602684 and US20050123546.
`
`in the
`residue
`least one galactose
`IL-11Ro-binding proteins with at
`oligosaccharide attached to the Fe region are also contemplated. Such proteins may
`have improved CDC function. Such proteins are described, e.g., in WO1997/30087 and
`WO01999/22764.
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`IL-11Roa-binding proteins can also comprise a Fe region capable of inducing
`enhanced levels of CDC. For example, hybrids of IgG1 and IgG3 produce antibodies
`having enhanced CDCactivity (Natsumeet al., Cancer Res. 68: 3863-3872, 2008).
`IL-11Ro-binding proteins can also or alternatively be fused to or conjugated to
`proteins (e.g., antibody variable regions) that bind to immuneeffector cells, e.g., by
`virtue of binding to CD3 or CD16.
`In one example,
`Methods for determining effector function are knownin the art.
`the level of ADCC activity is assessed using a Cr release assay, an europium release
`assay or a 5S release assay.
`In each of these assays, cells expressing IL-11Ra are
`cultured with one or more ofthe recited compounds for a time and under conditions
`sufficient for the compound to be taken up by the cell.
`In the case of a § release
`assay, the cells can be cultured with *5§-labeled methionine and/or cysteine for a time
`sufficient for the labeled amino acids to be incorporated into newly synthesized
`proteins. Cells are then cultured in the presence or absence of the IL-1 1Ra-binding
`protein and in the presence of immune effector cells, e.g., PBMCs and/or NK cells.
`The amount of ter, europium and/or 35§ in cell culture medium is then detected, and
`an increase in the presence of the protein compared to in the absence of protein
`indicates
`that
`the binding molecule/agent has
`effector
`function.
`Exemplary
`publications disclosing assays for assessing the level of ADCC induced by a protein
`include Hellstrom et al. Proc. Natl Acad. Sci. USA 8&3: 7059-7063, 1986 and
`
`Bruggemannet al., J. Exp. Med. 166: 1351-1361, 1987.
`Other assays for assessing the level of ADCC induced by a protein include
`ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc.
`CA, USA) or CytoTox 96® non-radioactive cytotoxicity assay (Promega, WI, USA).
`Alternatively, or additionally, effector function of an TL-11Ra-binding protein ts
`assessed by determining,
`its affinity for one or more FcyRs, e.g., as described in
`US7317091.
`
`Clq binding assays mayalso be carried out to confirm that the IL-1 1Ra-binding
`protein is able to bind Clq and may induce CDC. To assess complement activation, a
`CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol.
`Methods 202: 163, 1996).
`
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`
`Additional Modifications
`
`The present disclosure also contemplates additional modifications to an antibody
`or IL-11Ra-binding protein comprising an Fc region or constant region.
`
`35
`
`For example, the antibody comprises one or more amino acid substitutions that
`increase the half-life of the protein. For example, the antibody comprises a Fe region
`comprising one or more amino acid substitutions that increase the affinity of the Fe
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`region for the neonatal Fe region (FcRn). For example, the Fe region has increased
`affinity for FcRn at lower pH,e.g., about pH 6.0, to facilitate Fe/FeRn binding in an
`endosome. In one example, the Fc region has increased affinity for FcRn at about pH 6
`
`comparedto its affinity at about pH 7.4, whichfacilitates the re-release of Fc into blood
`following cellular recycling. These amino acid substitutions are useful for extending
`the half life of a protcin, by reducing clearance from the blood.
`
`Exemplary amino acid substitutions include T250Q and/or M428L or T252A,
`T2548 and T266F or M252Y, $254T and T256E or H433K and N434F according to
`the EU numbering system. Additional or alternative amino acid substitutions arc
`
`described, for example, in US20070135620 or US7083784.
`
`Exemplary IL-1 1Ra-Binding Proteins
`Exemplary variable region containing [L-11Ra-binding proteins produced by
`the inventors are described in Table 1.
`
`10
`
`15
`
`Table 1: Sequences of exemplary IL-11Ro-binding proteins
`
`
`
`Antibody Name V_ amino acid|Vy amino acid
`
` 5
`
`SEQ ID NO
`37
`37
`37
`
`SEQ ID NO
`
`
`
` 37
`
`
`
`37
`37
`37
`37
`
`37
`
`37
`37
`37
`
`37
`37
`37
`
` wmIN[DA[ATR[WINJe1O
`
`TS-17
`
`22
`
`37
`
`37
`
`37
`
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`

`
`
`46
`TS-108
`5
`
`47
`
`
`
`48
`
`49
`
`50
`TS-134
`5
`
`31
`
`wa No
`
`53
`
`34
`
`55
`
`TS-135
`
`TS-136
`
`TS-140
`
`TS-143
`
`TS-151
`
`A[a
`
`5
`
`5
`
`5
`
`WO 2014/121325
`
`~~
`
`
`19
`TS-20
`
`20
`TS-21
`
`21
`TS-22
`
`22
`
`TS-29
`
`Notd
`TS-32
`
`24
`TS-49
`
`25
`TS-51
`
`26
`TS-55
`
`No
`iw)oo)
`TS-58
`
`29
`TS-63
`
`27
`
`TS-57
`
`30
`
`TS-64
`
`a =
`TS-66
`
`32
`TS-69
`5
`
`33
`TS-71
`5
`
`34
`TS-76
`5
`
`35
`
`TS-79
`
`5
`
`w On
`TS-82
`
`37__|
`TS-88
`5
`
`41 ATnTN
`
`BRw|N
`
`TS-101
`
`TS-103
`
`44
`TS-104
`5
`
`45
`TS-107
`5
`
`
`38|TS-89 5
`
`39
`
`40
`
`
`
`
`
`PCT/AU2014/000083
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`
`
`
`Protein Production
`
`In one example, an IL-11Ra-binding protein described herein according to any
`example is produced by culturing a hybridoma under conditions sufficient to produce
`the protein, e.g., as described herein and/oras is knownintheart.
`
`10
`
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`
`Recombinant Expression
`
`In another example, an IL-11Ra-binding protein described herein according to
`
`any example is recombinant.
`In the case of a recombinant protein, nucleic acid encoding same can be cloned
`into expression constructs or vectors, which are then transfected into host cells, such as
`
`E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells,
`Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or
`mycloma cclls that do not otherwise produce the protcin. Exemplary cells used for
`
`expressing a protein are CHO cells, myeloma cells or HEK cells. Molecular cloning
`techniques to achieve these ends are knownin the art and described, for example in
`Ausubel e/ al.,
`(editors), Current Protocols in Molecular Biology, Greene Pub.
`Associates and Wiley-Interscience (1988,
`including all updates until present) or
`
`Sambrook ef al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
`Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods
`are suitable for the construction of recombinant nucleic acids. Methods of producing
`
`recombinant antibodies are also knownin theart, see, e.g., US4816567 or US5530101.
`Following isolation, the nucleic acid is inserted operably linked to a promoter in
`an expression construct or expression vector for further cloning (amplification of the
`DNA)or for expression in a cell-free system or in cells.
`
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`As used herein, the term “promoter” is to be taken in its broadest context and
`includes the transcriptional regulatory sequences of a genomic gene, including the
`TATA box or initiator element, which is required for accurate transcription initiation,
`
`with or without additional regulatory elements (e.g., upstream activating sequences,
`transcription factor binding sites, enhancers and silencers) that alter expression of a
`nucleic acid, ¢.g., in response to a developmental and/or external stimulus, or in a Ussuc
`
`specific manner. In the present context, the term “‘promoter’’ is also used to describe a
`recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or
`enhances the expression of a nucleic acid to which it is operably linked. Exemplary
`
`10
`
`promoters can contain additional copies of one or more specific regulatory elements to
`further enhance expression and/or alter
`the spatial expression and/or
`temporal
`expression of said nucleic acid.
`
`As used herein, the term “operably linked to" means positioning a promoter
`
`relative to a nucleic acid such that expression of the nucleic acid is controlled by the
`
`15
`
`promoter.
`
`Many vectors for expression in cells are available. The vector components
`
`limited to, one or more of the following: a signal
`generally include, but are not
`sequence, a sequence encoding a protein (c.g., derived from the information provided
`herein), an enhancer element, a promoter, and a transcription termination sequence. The
`
`skilled artisan will be aware of suitable sequences for expression of a protein.
`Exemplary signal sequencesinclude prokaryotic secretion signals (c.g., pelB, alkaline
`phosphatase, penicillinase, Ipp, or heat-stable enterotoxin IJ), yeast secretion signals
`(e.g.,
`invertase leader, a factor leader, or acid phosphatase leader) or mammalian
`secretion signals (c.g., herpes simplex gD signal).
`include cytomegalovirus
`Exemplary promoters active in mammalian cells
`immediate early promoter (CMV-IE), human elongation factor 1-@ promoter (EF1),
`small nuclear RNA promoters (Ula and U1/b), a-myosin heavy chain promoter, Simian
`virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus majorlate
`promoter, B-actin promoter; hybrid regulatory element comprising a CMV enhancer/ B-
`actin promoter or an immunoglobulin promoter or active fragment thereof. Examples of
`useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40
`(COS-7, ATCC CRL 1651); human embryonic kidneyline (293 or 293 cells subcloned
`for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or
`Chinese hamster ovary cells (CHO).
`
`Typical promoters suitable for expression in yeast cells such as for example a
`yeast cell selected from the group comprising Pichia pasioris, Saccharomyces
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`cerevisiae and S. pombe, include, but are not limited to, the ADH/ promoter, the GAL/
`promoter,
`the GAL4 promoter,
`the CUP/ promoter,
`the PHOS promoter,
`the nmt
`promoter, the RPR/ promoter, or the TEF/ promoter.
`
`introducing the isolated nucleic acid or expression construct
`Means for
`comprising same into a cell for expression are known to those skilled in the art. The
`technique used for a given cell depends on the known successful techniques. Mcansfor
`
`introducing recombinant DNAinto cells include microinjection, transfection mediated
`by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine
`(Gibco, MD, USA)and/or cell fectin (Gibco, MD, USA), PEG-mediated DNA uptake,
`
`electroporation and microparticle bombardment such as by using DNA-coated tungsten
`or gold particles (Agracetus Inc., WI, USA) amongst others.
`The host cells used to produce the protcin may be cultured in a varicly of media,
`
`depending on the cell type used. Commercially available media such as Ham's FIO
`
`(Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and
`Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing
`mammalian cells. Media for culturing other cell types discussed herein are known in
`the art.
`
`Isolation of Proteins
`
`Methods for isolating a protein are knownin the art and/or described herein.
`Where an IL-1 1Ra-binding protein is secreted into culture medium, supernatants
`from such expression systems can be first concentrated using a commercially available
`protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltration
`unit. A protcase inhibitor such as PMSF maybe included in any of the foregoing stcps
`to inhibit proteolysis and antibiotics may be included to prevent
`the growth of
`adventitious contaminants. Alternatively, or additionally, supernatants can be filtered
`and/or
`separated from cells
`expressing the protein,
`e.g.
`using continuous
`centrifugation.
`The IL-11Ra-binding protein prepared from the cells can be purified using, for
`example,
`ion exchange, hydroxyapatite chromatography, hydrophobic interaction
`chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A
`affinity chromatography or protein G chromatography), or any combination of the
`foregoing. These methods are known in the art and described,
`for example in
`W0O99/57134 or Ed Harlow and David Lane (editors) Antibodies: A Laboratory
`
`10
`
`15
`
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`
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`
`Manual, Cold Spring Harbor Laboratory, (1988).
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`The skilled artisan will also be aware that a protein can be modified to include a
`tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine
`lag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) lag, or a
`
`FLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then
`purified using methods knownin theart, such as, affinity purification. For example, a
`protein comprising a hexa-his tag is purified by contacting a sample comprising the
`
`protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag
`immobilized on a solid or semi-solid support, washing the sample to remove unbound
`protein, and subsequently cluting the bound protein. Alternatively, or in addition a
`
`ligand orantibody that binds to a tag is used in an affinity purification method.
`
`Conjugates
`In one example, an IL-11Ra-binding protein of the present disclosure is
`
`conjugated to a compound. For example, the compound is selected from the group
`consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a
`toxin, a nucleic acid, a peptide, a protein, a compoundthat increases the half life of the
`IL-11Ra-binding protein in a subject and mixtures thereof.
`The other compound can be directly or indirectly bound to the IL-11Re-binding
`protein (e.g., can comprise a linker in the case of indirect binding). Examples of
`
`iodine-131, yttrium-90 or indium-111), a
`compounds include, a radioisotope (e.g.,
`detectable label (c.g., a Muorophore or a fluorescent nanocrystal or quantum dot), a
`therapeutic compound (e.g., a chemotherapeutic or an anti-inflammatory), a colloid
`(e.g., gold), a toxin (e.g., ricin or tetanus toxoid), a nucleic acid, a peptide (e.g., a
`scrum albumin binding peptide), a protcin (c.g., a protcin comprising an antigen
`binding domain of an antibody or serum albumin), a compoundthat increases the half
`life of the IL-11Ra-binding protein in a subject (e.g., polyethylene glycol or other water
`soluble polymer having this activity) and mixtures thereof. Exemplary compoundsthat
`can be conjugated to a IL-11Re-binding protein of the disclosure and methods for such
`conjugation are known in the art and described, for example, in WO2010/059821.
`The IL-11Ra-binding protein may be conjugated to nanoparticles (for example
`reviewed in Kogan e/ al., Nanomedicine (Lond). 2: 287-306, 2007).
`The
`as
`nanoparticles may be metallic nanoparticles.
`The IL-11Ra-binding protein may be comprised in antibody-targeted bacterial
`minicells (for example as described in PCT/IB2005/000204).
`Some exemplary compounds that can be conjugated to a IL-11Ra-binding
`protein of the present disclosure are listed in Table 2.
`
`10
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`Table 2. Compounds useful in conjugation.
`
`(either
`Radioisotopes
`directly or indirectly)
`
` 84
`
`137 1257 107 S37. 157 "So, PAs : Sec, Oy By
`"Ru, Pd, 1™Rh, 1°! Rh, 1Sb, ?8Ba, THe, 711 At
`212,2p:
`153
`169
`oa
`2Pb 109pq Wy oy Soy
`Bi,
`Sm,
`Eu,
`IL
`13
`15
`18
`188
`16
`TF
`“Br, Br,Tc,
`''C,
`'°N,
`'°O,
`'8T,
`'88Re,
`Cu,
`“Br,
`203
`64,
`105
`198
`1
`177
`
`
`Pb, “Cu,1°Rh, ?8Au, Ag or 17’Lu
`
`67
`
`75
`
`Half life extenders
`
`Polyethylene glycol
`
`Fluorescent probes
`
`Biologics
`
`Glycerol
`Glucose
`
`Phycoerythrin (PE)
`Allophycocyanin (APC)
`Alexa Fluor 488
`
`Cy5.5
`
`fluorescent proteins such as Renilla luciferase, GFP
`immune modulators or proteins, such as cytokines,
`c.g., an interferon
`toxins
`
`an immunoglobulin or antibody or antibody variable
`region
`
`half life extenders
`
`such as albumin or antibody
`
`variable regions or peptides that bind to albumin
`
`Chemotherapeutics
`
`Taxol
`5-FU
`
`Doxorubicin
`
`Idarubicin
`
`Assaying Activity of an IL-11Ro-Binding Protein
`Binding to IL-11Ra and Mutants Thereof
`Tt will be apparent to the skilled artisan from the disclosure herein that some TL-
`11Ra-binding proteins of the present disclosure bind to the extracellular region (e.g., a
`region as described herein) of hIL-11Re and to specific mutant forms of extracellular
`region of hIL-11Ra (e.g., SEQ ID NO: 3 or SEQ ID NO: 85 without or with certain
`point mutations) and/or bind to both human and cynomolgus monkey IL-11Ra.
`
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`
`Methods for assessing binding to a protein are knowninthe art, e.g., as described in
`Scopes (Un: Protein purification: principles and practice, Third Edition, Springer
`Verlag, 1994). Such a method generally involves immobilizing the IL-11Ro-binding
`protein and contacting it with labeled antigen. Following washing to remove non-
`specific bound protein, the amount of label and, as a consequence, bound antigen is
`detected. Of course,
`the IL-11Roa-binding protein can be labeled and the antigen
`immobilized. Panning-type assays can also be used. Alternatively, or additionally,
`surface plasmon resonanceassays can be used.
`The assays described above can also be used to detect the level of binding of a
`protein to hIL-11Re or an extracellular region thereof (e.g., as contained within SEQ
`ID NO: 3) or to a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 85 or mutant form
`thereof.
`
`10
`
`In one example, an IL-11Ra-binding protein of the present disclosure binds to a
`
`polypeptide of SEQ ID NO: 95 at a level at least about 1.5 fold or 2 fold or 5 fold or 10
`fold or 50 fold or 100 fold or 150 fold or 160 fold or 200 fold lower than it binds to a
`
`15
`
`polypeptide of SEQ ID NO: 85.
`
`In one example, a protein of the present disclosure binds to a polypeptide of
`SEQ ID NO: 96 at a level at least about 1.5 fold or 2 fold or 5 fold or 10 fold or 50 fold
`
`or 100 fold or 150 fold or 160 fold or 200 fold lowerthan it binds to a polypeptide of
`
`SEQ ID NO: 85.
`
`In one cxample, a protein of the present disclosure binds to a polypeptide of
`SEQ ID NO: 86 at a level at least about 1.5 fold or 2 fold or 5 fold or 10 fold or 50 fold
`
`or 100 fold or 150 fold or 160 fold or 200 fold lower than it binds to a polypeptide of
`SEQ ID NO: 85.
`
`In one example, a protein of the present disclosure binds to a polypeptide of
`SEQ ID NO: 89 at a level at least about 1.5 fold or 2 fold or 5 fold or 10 fold or 50 fold
`
`or 100 fold or 150 fold or 160 fold or 200 fold lower than it binds to a polypeptide of
`SEQ ID NO: 85.
`
`The level of binding is conveniently determined using a biosensor.
`The present disclosure contemplates
`any combination of
`the foregoing
`characteristics.
`In one example, a protein described herein has all of the binding
`characteristics set forth in the preceding five paragraphs.
`
`Epitope Mapping
`
`In another example, the epitope bound by a protein described herein is mapped.
`Epitope mapping methods will be apparent to the skilled artisan. For example, a series
`
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`
`of overlapping peptides spanning the 1L-11Ra sequence or a region thereof comprising
`an epitope of interest, e.g., peptides comprising 10-15 amino acids are produced. The
`IL-11Ro-binding protein is then contacted to each peptide and the peptide(s) to whichit
`binds determined. This permits determination of peptide(s) comprising the epitope to
`which the protein binds.
`If multiple non-contiguous peptides are bound by the protein,
`the protein may bind a conformational cpitope.
`Alternatively, or in addition, amino acid residues within IL-11Ro are mutated,
`e.g., by alanine scanning mutagenesis or substitution of evolutionarily conserved amino
`acids, and mutations that reduce or prevent binding of the IL-11Ra-binding protein are
`determined. Any mutation that reduces or prevents binding of the IL-11Ra-binding
`protein is likely to be within the epitope boundbythe IL-1 1Ra-binding protein.
`Tn this regard, as shown herein, mutation of the valine at position 117 of TL-11R
`
`relative to SEQ ID NO:
`
`1| reduced or prevented binding of 8E2 and 8D10. Further
`
`testing of affinity matured variants of 8E2 confirmed that the V117 residue of IL-11R
`was more important for binding relative to the other residucs that were analyzed by
`mutation.
`
`Another method for determining a region comprising an epitope bound by an
`IL-11Roe-binding protein involved substituting a region of hIL-11Re with the
`corresponding region of a form of IL-11Ra to which the IL-11Ra-binding protein does
`not bind (e.g., mIL-11Ra). If the IL-11Ra-binding protein does not bind to the mutant
`form of IL-11Ra, then residues forming a part of the epitope of the protein may be
`within the substituted region.
`A further method for determining a region comprising an epitope involves
`binding IL-11Re or a region thereof to an immobilized IL-11Ra-binding protein of the
`present disclosure and digesting the resulting complex with proteases. Peptide that
`remains bound to the immobilized IL-11Ra-binding protein are then isolated and
`analyzed, e.g., using mass spectrometry, to determine their sequence.
`A further method involves converting hydrogens in IL-11Ra or a region thereof
`to deutrons and binding the resulting protein to an immobilized IL-11Ra-binding
`protein of the present disclosure. The deutrons are then converted back to hydrogen,
`the IL-11Ra or region thereof isolated, digested with enzymes and analyzed, e.g., using
`ma