WO 2014/121325
`
`PCT/AU2014/000083
`
`73
`
`effector function of an Fc region containing protein are known in the art and/or
`described herein.
`
`In one example, the Fc region is an IgG4 Fc region (i.e., from an IgG4 constant
`
`region), e.g., a human IgG4 Fc region. Sequences of suitable IgG4 Fc 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 exchange or 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 (half-molecule) 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 exchange occurs naturally in vivo and can be induced in vitm 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 at (1].,
`
`Sequences of Proteins of Immunological
`
`Interest Washington DC United States
`
`Department of Health and Human Seivices, 1987 and/or 1991).
`
`This position
`
`corresponds to position 228 of the hinge region according to the EU numbering system
`
`(Kabat er al., Sequences of Proteins of Immunological Interest Washington DC United
`
`States Department of Health and Human Services, 2001 and Edelman et 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 Pr0243 of human IgG1
`
`according to the numbering system of Kabat. Hinge regions of other IgG isotypes may
`
`be aligned with the IgGl sequence by placing the first and last cysteine residues
`
`forming inter—heavy chain disulphide (S—S) bonds in the same positions (see for
`
`example WO20lO/080538).
`
`10
`
`15
`
`25
`
`3O
`
`35
`
`Ex. 2001 - Page982
`Ex. 2001 - P396982
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`74
`
`Additional examples of stabilized lgG4 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 leueine
`
`(e.g., as described in WO2006/O33386). 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 leucine 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).
`
`10
`
`15
`
`In another example, the Fc region is a region modified to have reduced effector
`
`function, i.e., a "non—immunostimulatory Fc region”. For example, the Fc region is an
`
`IgGl Fc region comprising a substitution at one or more positions selected from the
`
`group consisting of 268, 309, 330 and 331.
`
`In another example, the Fc region is an
`
`IgGl Fc 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,
`
`A3305 and P3315 (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J
`
`Biol Chem. 276(9):659l—604, 2001). Additional examples of non—immunostimulatory
`
`Fc regions are described, for example, in Dall'Acqua et (11., J lmmzmol. 177 : 1129-1138
`
`2006; and/or I-Iezareh J Vim] ;75: 12161-12168, 2001).
`
`In another example, the Fc region is a chimeric Fc region, e.g., comprising at
`
`least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an
`
`IgGl antibody, wherein the Fc region comprises a substitution at one 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 WO20lO/O85682).
`
`Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 3071’,
`
`25
`
`309K, 309M, 309P, 323F, 3993, and 427F.
`
`Enhancing Effector Function
`
`In one example, an lL-llRa-binding protein of the present disclosure may
`induce effector function or enhanced effector function.
`
`30
`
`In the context of the present disclosure, “effector functions” refer to those
`
`biological activities mediated by cells or proteins that bind to the Fc region (a native
`
`sequence Fc region or amino acid sequence variant Fc region) of an antibody that result
`
`in killing of a cell. Examples of effector functions induced by antibodies include:
`
`complement dependent cytotoxicity; antibody-dependent-cell—mediated cytotoxieity
`
`(ADCC); antibody-dependent—cell-phagocytosis (ADCP); and B-cell activation.
`
`Ex. 2001 - Page983
`Ex. 2001 - PageQS3
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`75
`
`In one example, an IL-l lRa-binding protein of the present disclosure binds to
`
`IL-11R0t 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 IL-1 1Rot-binding protein remains bound to the IL-1 1R0t 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-l 1Ra-binding protein of the present disclosure is capable
`
`of inducing enhanced effector function, e.g., by virtue of a modified Fc 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 F c region. Enhancing effector function induced by a IL-l lRa-
`
`binding protein of the disclosure may result in enhanced therapeutic or prophylactic
`
`effects, e. g., by not only blocking the action of IL-1 lRot but also by killing or depleting
`
`cells causing a condition, e.g., by killing auto-reactive T cells.
`
`In one example, the Fc region of an IL-l 1R0t-binding protein of the disclosure is
`
`modified to increase the level of effector function it is capable of inducing compared to
`
`the Fc 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 number of 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 Fc
`
`region binds With greater affinity to one or more FcyRs, such as FcyRIII.
`
`In one
`
`example,
`
`the Fc 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 amino acid substitutions S239D/I332E, numbered according to the EU index
`
`of Kabat. This F c region has about 14 fold increase in affinity for FevRIIIa compared
`
`to a wild-type Fc region and about 3.3 increased ability to induce ADCC compared to a
`
`wild—type Fc region. In one example, the Ec region comprises the following amino acid
`
`substitutions $239D/A330L/I332E, numbered according to the EU index of Kabat.
`
`This Fc region has about 138 fold increase in affinity for F cyRIIIa compared to a Wild-
`
`type Fc region and about 323 fold increased ability to induce ADCC compared to a
`
`wild—type Fc region.
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`Ex. 2001 - Page984
`Ex. 2001 - P396984
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`76
`
`Additional amino acid substitutions that increase ability of a Fc region to induce
`
`effector function are known in the art and/or described, for example, in US6737056 or
`US73 17091.
`
`In one example, the glycosylation of the Fc region is altered to increase its
`
`ability to induce enhanced effector function.
`
`In this regard, native antibodies produced
`
`by mammalian cells typically comprise a branched, biantennary oligosaccharide that is
`
`generally attached by an N—linkage to Asn297 of the CH2 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
`
`GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some examples,
`
`Fc regions according to the present disclosure comprise a carbohydrate structure that
`
`lacks fucose attached (directly or indirectly) to an Fc region,
`
`i.e., the Fc region is
`
`“afucosylated”. Such variants may have an improved ability to induce ADCC. Methods
`
`for producing afucosylated antibodies include, expressing the antibody or antigen
`
`binding fragment thereof in a cell line incapable of expressing u-1,6-fucosyltransferase
`
`(FUTS) (e.g., as described in Yumane—Ohnuki et 61]., Biotechnol. Bioengineer. 87: 614-
`
`622, 2004), expressing the antibody or antigen binding fragment
`
`thereof in cells
`
`expressing a small interfering RNA against FUTS (e.g., as described in Mori et (11.,
`
`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—3 10, 2007). The present disclosure also contemplates the use of
`
`proteins having a reduced level of fucosylation, e.g., produced using a cell
`
`line
`
`10
`
`15
`
`20
`
`25
`
`modified to express B—(1,4)—N—acetylglucosaminyltransferase III (GnT-III) (e.g., as
`described in Umana et al., Nat. Biotechnol. 77: l76-180, l999).
`
`Other methods include the use of cell lines which inherently produce antibodies
`
`capable of inducing enhanced Fc-mediated effector function (e.g. duck embryonic
`
`derived stem cells for the production of viral vaccines, W02008/129058; Recombinant
`
`protein production in avian EBX® cells, WC 2008/ 142124).
`
`IL-llRa-binding proteins of the present disclosure also include those with
`
`bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the
`
`Fc region is bisected by GlcNAc. Such proteins may have reduced fucosylation and/or
`
`improved ADCC function. Examples of
`US6602684 and US20050123546.
`
`such proteins are described, e.g.,
`
`in
`
`IL-l lRu-binding proteins with at
`
`least one galactose
`
`residue
`
`in the
`
`oligosaccharide attached to the Fc region are also contemplated. Such proteins may
`
`have improved CDC function. Such proteins are described, e.g., in WOl997/30087 and
`WOl999/22764.
`
`30
`
`35
`
`Ex. 2001 - Page985
`Ex. 2001 - PageQSS
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`77
`
`lL-l lRtx-binding proteins can also comprise a Fc region capable of inducing
`
`enhanced levels of CDC. For example, hybrids of IgG1 and IgG3 produce antibodies
`
`having enhanced CDC activity (Natsume er al., Cancer Res. 68: 3863-3872, 2008).
`
`IL-l 1R0t-binding proteins can also or alternatively be fiised to or conjugated to
`
`proteins (e.g., antibody variable regions) that bind to immune effector cells, e.g., by
`
`virtue of binding to CD3 or CD16.
`
`Methods for determining effector function are known in the art.
`In one example,
`the level of ADCC activity is assessed using a 51Cr release assay, an europium release
`assay or a 358 release assay.
`In each of these assays, cells expressing IL-llROt are
`cultured with one or more of the recited compounds for a time and under conditions
`sufficient for the compound to be taken up by the cell.
`In the case of a 353 release
`assay, the cells can be cultured with 35S-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 lL-l lRa-binding
`
`protein and in the presence of immune effector cells, e.g., PBMCs and/or NK cells.
`The amount of 51Cr, europium and/or 353 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 83: 7059—7063, 1986 and
`
`Bruggemann et al., J. Exp. Med. 166: 1351—1361, 1987.
`
`Other assays for assessing the level of ADCC induced by a protein include
`
`ACTITM 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 lL-l lRa-binding protein is
`
`assessed by determining its affinity for one or more FcyRs, e.g., as described in
`US73l7091.
`
`Clq binding assays may also be carried out to confirm that the IL-1 lRa-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 (11., J. Immzmol.
`Methods 202: 163, 1996).
`
`Additional Modifications
`
`The present disclosure also contemplates additional modifications to an antibody
`
`or lL-l lRot-binding protein comprising an F c region or constant region.
`
`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 Fc region
`
`comprising one or more amino acid substitutions that increase the affinity of the Fc
`
`10
`
`20
`
`25
`
`3O
`
`35
`
`Ex. 2001 - Page986
`Ex. 2001 - P396986
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`78
`
`region for the neonatal Fc region (FcRn). For example, the Fc region has increased
`
`affinity for FcRn at lower pH, e.g., about pH 6.0, to facilitate Ec/EcRn binding in an
`
`endosome. In one example, the Fc region has increased affinity for FcRn at about pH 6
`
`compared to its affinity at about pH 7.4, which facilitates the re—release of PC into blood
`
`following cellular recycling. These amino acid substitutions are useful for extending
`
`the half life of a protein, by reducing clearance from the blood.
`
`Exemplary amino acid substitutions include T250Q and/or M428L or T252A,
`
`T2545 and T266F or M252Y, S254T and T256E or H433K and N434F according to
`
`the EU numbering system. Additional or alternative amino acid substitutions are
`
`described, for example, in U520070l35620 or US7083784.
`
`Exemplary IL-I 1Ra-Binding Proteins
`
`Exemplary variable region containing lL-l lRa-binding proteins produced by
`the inventors are described in Table 1.
`
`Table 1: Sequences of exemplary lL-l lRa-binding proteins
`
`10
`
`15
`
`Antibody Name
`
`VL amino acid VH amino acid
`
` l
`
`SEQ ID NO
`SEQ ID NO
`37
`.
`8E2
`to
`37
`37
`
`somuom-hw
`
`37
`37
`37
`37
`
`37
`
`37
`
`
`
` 37
`
`
`
`37
`
`37
`
`37
`
`C7J\]O\'Jl-I>UJNt—O
`
`37
`37
`37
`
`37
`
`TS—l7
`22
`37
`
`Ex. 2001 - Page987
`Ex. 2001 - PageQS7
`
`

`

`WO 2014/121325
`
`PC T/AU20 14/000083
`
`79
`
`
`19
`T320
`23
`37
`
`20 mm
`24
`37
`
`21
`T522
`25
`37
`
`22
`
`18-29
`
`26
`
`37
`
`33
`37
`
`24
`TS-49
`28
`37
`
`25
`TS-51
`29
`37
`
`26
`TS—55
`30
`37
`
`27
`
`TS-57
`
`31
`
`37
`
`33
`37
`
`29
`TS—63
`33
`37
`
`30
`
`TS-64
`
`34
`
`37
`
`31
`33
`
`32
`TS-69
`5
`39
`
`33
`TS-71
`5
`4o
`
`34
`TS—76
`5
`41
`
`35
`
`TS-79
`
`5
`
`42
`
`36
`43
`
`37
`TS—88
`5
`44
`
`45
`5
`TS-89
`38
`4;C\
` U1
`
`40
`5
`47
`
`
`41
`5
`48
`
`
`43
`
`TS-103
`
`5
`
`50
`
`44
`TS-1()4
`5
`51
`
`45
`TS—107
`5
`52
`
`46
`TS-108
`5
`53
`
`47
`5
`
`48
`
`5
`
`
`
`
`
`
`
`
`
`49
`5
`
`50
`TS—134
`5
`57
`
`51
`
`33
`
`53
`
`54
`
`TS-135
`
`TS—l40
`
`TS-143
`
`5
`
`5
`
`5
`
`58
`
`39
`
`6O
`
`61
`
`55
`
`TS—151
`
`5
`
`62
`
`Ex. 2001 - Page988
`Ex. 2001 - PageQSS
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`
`
`80
`
`
`
`
`
`
`
`
`Protein Production
`
`In one example, an IL-l lRa-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/or as is known in the art.
`
`10
`
`15
`
`20
`
`Recombinant Expression
`
`In another example, an IL-l lRot-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
`
`myeloma cells that do not otherwise produce the protein. Exemplary cells used for
`
`expressing a protein are CHO cells, myeloma cells or HEK cells. Molecular cloning
`
`techniques to achieve these ends are known in the art and described, for example in
`
`Ausubel el al.,
`
`(editors), Current Protocols in Molecular Biology, Greene Pub.
`
`Associates and Wiley—lnterscience (1988,
`
`including all updates until present) or
`
`Sambrook et
`
`(11., 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 known in the art, see, e.g., US4816567 or USSS30lOl.
`
`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.
`
`Ex. 2001 - Page989
`Ex. 2001 - PageQSQ
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`81
`
`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, e.g., in response to a developmental and/or external stimulus, or in a tissue
`
`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
`
`l5
`
`promoter.
`
`Many vectors for expression in cells are available. The vector components
`
`generally include, but are not
`
`limited to, one or more of the following: a signal
`
`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 sequences include prokaryotic secretion signals (c.g., pelB, alkaline
`
`phosphatase, penicillinase, Ipp, or heat—stable enterotoxin II), yeast secretion signals
`
`(e.g.,
`
`invertase leader, a factor leader, or acid phosphatase leader) or mammalian
`
`secretion signals (c.g., herpes simplex gD signal).
`
`Exemplary promoters active in mammalian cells
`
`include cytomegalovinis
`
`immediate early promoter (CMV—IE), human elongation factor l—oc promoter (EFl),
`
`small nuclear RNA promoters (Ula and Ulb), oc—myosin heavy chain promoter, Simian
`
`virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late
`
`promoter, B-actin promoter; hybrid regulatory element comprising a CMV enhancer/ [3-
`
`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 kidney line (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 pastoris, Saccharomyces
`
`25
`
`3O
`
`35
`
`Ex. 2001 - Page990
`Ex. 2001 - PageQQO
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`82
`
`cerevisiae and S. pombe, include, but are not limited to, the ADHI promoter, the GAL]
`
`promoter,
`
`the GAL4 promoter,
`
`the CUP] promoter,
`
`the PH05 promoter,
`
`the nmt
`
`promoter, the RPR] promoter, or the TEF1 promoter.
`
`Means for
`
`introducing the isolated nucleic acid or expression construct
`
`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. Means for
`
`introducing recombinant DNA into cells include microinjection, transfection mediated
`
`by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine
`
`(Gibco, MD, USA) and/or cellfectin (Gibeo, 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 protein may be cultured in a variety of media,
`
`depending on the cell type used. Commercially available media such as Ham's F10
`
`(Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMl-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 QfProteins
`
`Methods for isolating a protein are known in the art and/or described herein.
`
`Where an lL-l lRa-binding protein is secreted into culture medium, supernatants
`
`from such expression systems can be first concentrated using a commercially available
`
`protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration
`
`unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps
`
`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 lL-l lRa-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
`
`WO99/57l34 or Ed Harlow and David Lane (editors) Antibodies: A Laboratory
`
`Manual, Cold Spring Harbor Laboratory, (1988).
`
`10
`
`15
`
`25
`
`3O
`
`35
`
`Ex. 2001 - Page991
`Ex. 2001 - P396991
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`83
`
`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
`
`tag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a
`
`FLAG tag, or a glutathione S—transferase (GST) tag. The resulting protein is then
`
`purified using methods known in the art, 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 eluting the bound protein. Alternatively, or in addition a
`
`ligand or antibody that binds to a tag is used in an affinity purification method.
`
`Conjugates
`
`In one example, an IL-l lRa-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, 3 protein, a compound that increases the half life of the
`
`lL- 1 lRo-binding protein in a subject and mixtures thereof.
`
`The other compound can be directly or indirectly bound to the lL-l lRa-binding
`
`protein (e.g., can comprise a linker in the case of indirect binding). Examples of
`
`compounds include, a radioisotope (e.g.,
`
`iodine—131, yttrium—9O or indium—lll), a
`
`detectable label (c.g., a fluorophorc or a fluorescent nanocrystal or quantum dot), a
`
`therapeutic compound (c.g., a chemotherapeutic or an anti—inflammatmy), a colloid
`
`(e.g., gold), a toxin (e.g., ricin or tetanus toxoid), a nucleic acid, a peptide (e.g., a
`
`serum albumin binding pcptidc), a protein (c.g., a protein comprising an antigen
`
`binding domain of an antibody or serum albumin), a compound that increases the half
`
`life of the lL- l lRa-binding protein in a subject (c.g., polyethylene glycol or other water
`
`soluble polymer having this activity) and mixtures thereof. Exemplary compounds that
`
`can be conjugated to a IL-l lRa-binding protein of the disclosure and methods for such
`
`conjugation are known in the art and described, for example, in WOZOlO/OS9821.
`
`The lL-l lRu-binding protein may be conjugated to nanoparticles (for example
`
`as
`
`reviewed in Kogan et al., Nanomedicine (Land). 2: 287—306, 2007).
`
`The
`
`nanoparticles may be metallic nanoparticles.
`
`The lL-llRu-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 lL-l lRa-binding
`
`protein of the present disclosure are listed in Table 2.
`
`10
`
`15
`
`25
`
`3O
`
`35
`
`Ex. 2001 - Page992
`Ex. 2001 - P396992
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`84
`
`Table 2. Corn ounds useful in con‘uation.
`
`Radioisotopes
`
`(either
`
`123I, 125I, 1301, 133I, 1351, 4780, 72As , 7280, 90Y, 88Y,
`
`directly or indirectly)
`
`97Ru, 100Pd, IOImRh, 101mRh, 1195b, 12Ee’Ba, 197l-lg, 211At,
`
`ZlZBi,
`
`lSism’ 169E119 iisz, UNPd’
`
`'“In 5 67611, “Gu,
`
`6701. 75Br, 76Br 9 77313 991nTC’ ”C, lSN’ 150’ 18L 188RC’
`
`203Pb, 64cm IOSRh’ 198Au’ 199A or 177Lu
`
`Half life extenders
`
`Polyethylene glycol
`
`Glycerol
`Glucose
`
`Fluorescent probes
`
`Phycoerythrin (PE)
`
`Allophyeoeyanin (APC)
`Alexa Fluor 488
`
`C 5.5
`
`
`
`Biologics
`
`fluorescent proteins such as Renilla luciferase, GFP
`
`immune modulators or proteins, such as cytokines,
`
`e.g., an interferon
`toxins
`
`an immunoglobulin or antibody or antibody variable
`
`region
`
`half life extenders
`
`such as albumin or antibody
`
`variable re ions or ne ntides that bind to albumin
`
`Chemotherapeutics
`
`Taxol
`5 -FU
`
`Doxorubiein
`
`ldarubicin
`
`Assaying Activity of an IL-llRa—Binding Protein
`
`Binding [0 IL—11Ra and iMuz‘anz‘s Thereof
`
`It will be apparent to the skilled artisan from the disclosure herein that some IL-
`
`llRa-binding proteins of the present disclosure bind to the extracellular region (e. g., a
`
`region as described herein) of hlL-l lRa and to specific mutant forms of extracellular
`
`region of hlL-l lRa (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 lL-l lRa.
`
`Ex. 2001 - Page993
`Ex. 2001 - PageQQ3
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`85
`
`Methods for assessing binding to a protein are known in the alt, e.g., as described in
`
`Scopes (In: Protein purification: principles and practice, Third Edition, Springer
`
`Verlag, 1994). Such a method generally involves immobilizing the IL-1 lRot-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-llRot-binding protein can be labeled and the antigen
`
`immobilized. Panning—type assays can also be used. Alternatively, or additionally,
`
`surface plasmon resonance assays can be used.
`
`The assays described above can also be used to detect the level of binding of a
`
`10
`
`protein to hIL-llRa 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.
`
`In one example, an IL-l 1Ra-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 lOO 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 l0 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: 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
`
`25
`
`3O
`
`35
`
`Ex. 2001 - Page994
`Ex. 2001 - P396994
`
`

`

`WO 2014/121325
`
`PCT/AU2014/000083
`
`86
`
`of overlapping peptides spanning the lL-l lRa sequence or a region thereof comprising
`
`an epitope of interest, e.g., peptides comprising 10-15 amino acids are produced. The
`
`IL- 1 lRot-binding protein is then contacted to each peptide and the peptide(s) to which it
`
`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 epitope.
`
`Alternatively, or in addition, amino acid residues within IL-l lRa are mutated,
`
`e.g., by alanine scanning mutagenesis or substitution of evolutionarily conserved amino
`
`acids, and mutations that reduce or prevent binding of the lL-l l Rot-binding protein are
`
`determined. Any mutation that reduces or prevents binding of the IL-1 lRa-binding
`
`protein is likely to be within the epitope bound by the IL-1 lRa-binding protein.
`
`in this regard, as shown herein, mutation of the valine at position 1 17 of lL-l lR
`
`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-llR
`
`was more important for binding relative to the other residues that were analyzed by
`mutation.
`
`Another method for determining a region comprising an epitope bound by an
`
`lL-l l Rot-binding protein involved substituting a region of hlL-l lRot with the
`
`corresponding region of a form of IL-1 lRa to which the IL-1 lRa-binding protein does
`
`not bind (e.g., mIL-l lRa). If the lL-llRa-binding protein does not bind to the mutant
`
`form of IL-1 lRa, 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 lL-l lRot or a region thereof to an immobilized lL-l lRu-binding protein of the
`
`pres