WO2013075027A2 - Anti-sil6xr complex binding domains and methods of use - Google Patents

Abstract

The present invention relates generally to fusion proteins comprising anti-sIL6xR binding domains and methods of using same. Such proteins are useful, for example, in methods for treating any of a variety of inflammatory disorders including rheumatoid arthritis, psoriasis and ulcerative colitis.

Description

ANTI-SIL6XR COMPLEX BINDING DOMAINS AND METHODS OF USE

This application claims priority to U.S. Provisional Patent Application No. 61/561 ,108, filed November 17, 201 1 , the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is EMER_018_01 WO_ST25.txt. The text file is 381 KB, was created on

November 16, 2012, and is being submitted electronically via EFS-Web.

BACKGROUND

Technical Field

The present invention relates generally to polypeptides comprising anti- slL6xR binding domains and methods of using same. Such proteins are useful, for example, in methods for treating any of a variety of disorders including inflammatory disorders such as rheumatoid arthritis, psoriasis and colitis.

Description of the Related Art

Interleukin 6 ("IL6") is a pleiotropic cytokine that regulates host immune responses, inflammation, hematopoiesis, and oncogenesis. IL6 biology is mediated by a multicomponent molecular system with two distinct modes of signaling operative on overlapping but non-identical cell populations. These are referred to as cis-signaling (also known as "classical" signaling) and trans- signaling.

In cis-signaling, IL6 binds to cell surface IL6 receptor, the ligand binding part of IL6R that is referred to as IL6Ra or CD126 (previously called gp80). The cell-bound IL6/IL6Ra complex in turn binds to non-ligand binding but signal transducing membrane protein gp130 (also known as IL6ST or CD130), which induces gp130 dimerization and initiation of signaling. Thus, cis-signaling is restricted to the subset of cell types that express cell-surface IL6Ra, which is generally limited to, for example, mitogen-activated B cells, T cell subsets, peripheral monocytes, and certain tumors. The resultant ternary complex on the cell surface assembles into a very stable hexamer with a 2:2:2 ratio of

IL6:IL6Ra:gp130 (Boulanger et al. (2003) Science 300:2101 ).

Soluble forms of IL-6R are generated from proteolysis of membrane forms of IL-6R or from mRNA alternative splicing. In trans-signaling, soluble IL6Ra ("slL6R") complexes with IL6 and the resulting circulating slL6xR complex can bind to and activate any gp130-expressing cell (but not cells also expressing IL6R, Taga et al. (1989) Cell 58:573). Many, perhaps all or nearly all, cells in the human body express gp130. Because gp130 is ubiquitous, trans-signaling can affect many cell types and thereby sometimes cause disease.

The membrane protein gp130 also exists in soluble form ("sgp130"), which can bind slL6xR complex in circulation. But, the slL6xR complex binds equally well to membrane and soluble gp130 (see Jones et al., (2005) J.

Interferon Cytokine Res. 25:241 ). Therefore, a molar excess of sgp130 can inhibit trans-signaling (by reducing the amount of available slL6xR complex in circulation), which will not significantly affect cis-signaling because the affinity of sgp130 is orders of magnitude less, as compared to cell surface gp130, for cell- bound IL6/IL6Ra complex (see, e.g., Jostock et al. (2001 ) Eur. J. Biochem. 268:160). Thus, it has been suggested that sgp130 may be useful in inhibiting IL6 activity (see, e.g., Jostock et al. (2001 ) Eur. J. Biochem. 268:160). But, in addition to IL6, gp130 is a common signal-transducing protein for a family of gp130 cytokines. These include leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), neuropoietin (NP), cardiotropin like cytokine (CLC), oncostatin M (OSM), IL-27, IL-31 and cardiotrophin-1 (CT-1 ). Hence, although sgp130 can inhibit trans-signaling, administering such a compound to patients may have some unintended adverse effects.

Increased production of IL6 has been implicated in various disease processes, including Alzheimer's disease, autoimmunity {e.g., rheumatoid arthritis, SLE), inflammation, myocardial infarction, Paget's disease,

osteoporosis, solid tumors (e.g., colon cancer, RCC prostatic and bladder cancers), certain neurological cancers, B-cell malignancies, such as

Castleman's disease, some lymphoma subtypes, CLL, and, in particular, multiple myeloma. In some instances, IL-6 is implicated in proliferation pathways because it acts with other factors, such as heparin-binding epithelial growth factor and hepatocyte growth factor. Several IL6 and IL6Ra antibody antagonists are known. For example, for IL6, Way et al. (US Patent Application Publication No. 2007/0178098) disclose antibodies against IL6 to sterically block IL6 or slL6xR complex from binding to gp130 (see also US Patent No. 7,291 ,721 ). For example, for IL6Ra, Kishimoto (US Patent No. 5,670,373) discloses antibodies against IL6Ra that inhibit IL6 activity. There is a need in the art, however, for improved anti-IL6, anti-IL6R and anti-IL6xR therapeutics, particularly for treating patients who relapse or fail to respond to current treatment alternatives.

BRIEF SUMMARY

Some embodiments of the invention provide an isolated polypeptide comprising a binding domain that binds a soluble IL6/IL6R (slL6xR) complex, wherein the isolated polypeptide: (a) binds to the slL6xR complex with a higher affinity than either IL6 alone or IL6Ra alone; (b) competes with membrane gp130 for binding to the slL6xR complex; (c) binds to human native slL6xR about at least 5-fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2; and (d) the polypeptide is not a gp130.

Another aspect of the present disclosure provides an isolated

polypeptide comprising a binding domain that binds slL6xR wherein the binding domain comprises: (a) a VL region comprising a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ; or (b) a VH region comprising a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or (c) a VL of (a) and a VH of (b).

Some aspects of the invention provide an isolated polypeptide that binds to a slL6xR wherein the isolated polypeptide comprises from amino-terminus to carboxy-terminus: (a) a binding domain, wherein the binding domain binds to human native slL6xR about at least 5-fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2; (b) an immunoglobulin heavy chain CH2 constant region polypeptide, and (c) an immunoglobulin heavy chain CH3 constant region polypeptide. In some embodiments, an isolated

polypeptide comprises a hinge region between (a) and (b).

Some embodiments of the invention provide an isolated polypeptide comprising the structure N-BD1 -L1 -CH2CH3-L2-BD2-C wherein: N is the amino-terminus, C is the carboxy terminus, BD1 comprises a binding domain that binds slL6xR or a target molecule other than slL6xR; L1 is a first linker peptide; -CH2CH3- comprises an immunoglobulin CH2 and CH3 constant region; L2 is a second linker peptide; BD2 is a binding domain that specifically binds slL6xR or a target molecule other than slL6xR. In some embodiments, at least one of either BD1 or BD2, or both, is a binding domain that binds to human native slL6xR, e.g., about at least 5-fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2. In some embodiments, L1 is an immunoglobulin lgG1 hinge region having an amino acid sequence selected from the group consisting of SEQ ID NOs:120-192. In some embodiments, L2 is a linker peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS:193-360 and 745-748. In some embodiments, BD1 is a TNF antagonist or a TGF antagonist and BD2 is a binding domain that specifically binds human slL6xR complex. In some embodiments, BD2 is a TNF antagonist or a TGF antagonist and BD1 is a binding domain that specifically binds human slL6xR complex. In some embodiments, BD1 and/or BD2 comprises: (a) a VL region comprising a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ; or (b) a VH region comprising a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or (c) a VL of (a) and a VH of (b).

The invention also provides polypeptides contained in a first single chain polypeptide comprising a first heterodimerization domain that is capable of associating with a second single chain polypeptide comprising a second heterodimerization domain, wherein the first and second heterodimerization domain is different and wherein the associated first and second single chain polypeptides form a polypeptide heterodimer.

In some embodiments, a slL6xR binding domain comprises: (a) a VL region comprising a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ; or (b) a VH region comprising a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or (c) a VL of (a) and a VH of (b). In some embodiments, a VL CDR3 is SEQ ID NO:16 and a VH CDR3 is SEQ ID NO:15.

In some embodiments polypeptides of the invention comprise an Fc region constant domain; a hinge region disposed C-terminal to the Fc region constant domain; and a binding domain and in some embodiments the binding domain binds to human native slL6xR about at least 5-fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2.

Some aspects of the invention provide an isolated polypeptide comprising a binding domain which binds to a mutated Site III epitope of the IL- 6 signaling complex, wherein the mutated Site III epitope comprises at least mutations at one or two positions selected from F134, 1170 and R132 of the IL6R portion of the hyperlL6 fusion protein as set forth in SEQ ID NO:749.

In some embodiments, an isolated polypeptide binds to human native slL6xR about 5-25 fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2. In some embodiments, an isolated polypeptide or binding domain of the invention inhibits the biological activity of human native slL6xR complex. In some embodiments, an isolated polypeptide of the invention binds to IL6Ra alone with a higher affinity than to IL6 alone, preferentially inhibits IL6 trans- signaling over IL6 cis-signaling, inhibits the biological activity of a human native slL6xR complex, binds to human native slL6xR about at least 5-fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2 or any combination thereof. In some embodiments, inhibition of the biological activity of a human native slL6xR complex is statistically significantly greater as compared to an isolated polypeptide with an amino acid sequence consisting of SEQ ID NO:2. In some embodiments, biological activity is measured by cell proliferation and/or STAT3 phosphorylation induced by the human native slL6xR complex. In some instances, the cell proliferation comprises

proliferation of a BAF3 cell line expressing gp130. In some embodiments, an isolated polypeptide binds to human native slL6xR about at least 5-fold or 5-25 fold better in an ELISA than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2.

In some embodiments, polypeptides or fusion proteins described herein comprise a VL and/or VH region. In some embodiments, VL and VH regions are humanized. In some embodiments, a VL region comprises the amino acid sequence of SEQ ID NO:14 and/or a VH region comprises the amino acid sequence of SEQ ID NO:13.

In a further embodiment, the isolated polypeptide is an antibody or an antigen-binding fragment thereof, or a polypeptide comprising an antibody or antigen-binding fragment thereof. In another embodiment, the antibody or antigen-binding fragment thereof is non-human, chimeric, humanized or human. In one embodiment, an antibody or antigen-binding fragment thereof comprises a VL region comprising the amino acid sequence of any one of the sequences set forth in SEQ ID NOS:14, 20, 25 and 30. In an additional embodiment, an antibody or antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence of any one of the sequences set forth in SEQ ID NOS:13, 19, 24 and 29. In another embodiment, an antibody or antigen- binding fragment thereof comprises a VL region comprising the amino acid sequence as set forth in SEQ ID NO:14 and a VH region comprising the amino acid sequence as set forth in SEQ ID NO:13. In other embodiments, the isolated polypeptides or binding domains of the present disclosure may be in the form of or comprise a Fab fragment, an F(ab')2 fragment, an scFv, a dAb, and a Fv fragment and in some embodiments, an scFv comprises the amino acid sequence provided in amino acids 1 -247 of SEQ ID NO:12. In some embodiments, an scFv comprises a VL CDR1 amino acid sequence of SEQ ID NO:8, a VL CDR2 amino acid sequence of SEQ ID NO:9, a VL CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 , a VH CDR1 amino acid sequence of SEQ ID NO:5, a VH CDR2 amino acid sequence of SEQ ID NO:6, and a VH CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7.

In some embodiments, an isolated polypeptide may comprise a hinge region having an amino acid sequence of any one of SEQ ID NOS:37-70. In a further embodiment of an isolated polypeptide, CH2 and the CH3 domains comprise an immunoglobulin CH2 and a CH3 domain, e.g., of lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2 or IgD. In some embodiments, a human lgG1 CH2 domain may comprise the amino acid sequence of SEQ ID NO:96. In some embodiments, a human lgG1 CH3 domain may comprise the amino acid sequence of SEQ ID NO:436. Illustrative fusion polypeptides comprise an amino acid sequence selected from the sequences set forth in SEQ ID NOS:12, 18, 23 and 28. Some embodiments of the invention utilize a variant human CH2 and CH3 domain lacking one or more effector functions, e.g., derived from lgG1 .

A further aspect of the present disclosure provides a composition comprising a fusion polypeptide or an isolated polypeptide described herein and a pharmaceutically acceptable excipient. Another aspect of the disclosure provides an expression vector capable of expressing the fusion polypeptides or the isolated polypeptides described herein and also isolated host cells comprising the expression vector.

Another aspect of the present disclosure provides methods for treating an inflammatory disorder comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a fusion polypeptide or an isolated polypeptide described herein and a pharmaceutically acceptable excipient. In some embodiments, an inflammatory disorder may be selected from the group consisting of rheumatoid arthritis, psoriasis, colitis, ulcerative colitis, Crohn's disease, and cardiovascular disease.

Another aspect of the disclosure provides methods for treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a fusion polypeptide or an isolated polypeptide described herein and a pharmaceutically acceptable excipient. In some ebodiments, a cancer may be selected from the group consisting of colon cancer; ovarian cancer; pancreatic cancer; prostate cancer, non-Hodgkin's lymphoma, kidney cancer, lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A and 1 B are graphs from an ELISA showing that the A2 anti- human hyper IL-6 binding domain binds hyper IL-6 but does not bind human IL- 6.

Figure 2A and 2B depict the results of a TF-1 cell proliferation assay showing that the A2 SMIP specifically blocks hyperlL-6 (hHIL-6) induced TF-1 cell proliferation without inhibiting human IL-6 (hlL-6) function.

Figure 3A is a diagram of the Dyax phagemid pMID21 construct.

Reformatting the A2 binding domain into the phagemid pMID21 replaces the Fab and CH1 regions shown in Figure 3A resulting in the construct as shown in Figure 3B which was used in the phage display screening. Figure 3C depicts the amino acid sequence of the A2 scFv (VH-VL) binding domain as provided in SEQ ID NO: 32, with the VL and VH CDR sequences indicated in bold and underlined.

Figure 4A shows the results from binding studies for A2 mutant binding domains. Three binding domains (ATH105, ATH072, and ATH104) showed increased binding to hyper IL6 compared to A2, including ATH105 (A2M1 ). Figure 4B shows the amino acid sequence of the A2M1 mutant including the scFv binding domain (VH-VL) (SEQ ID NO:12). The VH and VL CDR3 are underlined with the mutated S7T and V10P positions shown with double underlining and bolded. The (G₄S)3 linker connecting the VH and VL regions is shown with a double underline.

Figure 5 depicts the results of a TF-1 cell proliferation assay showing that the affinity matured A2 SMIP mutants (ATH072, ATH104, ATH105 (A2M1 ), and ATH106) have enhanced blocking of hHIL-6 induced TF-1 proliferation compared to ATH064 (A2 SMIP).

Figure 6 shows that a mammalian in vitro system for expressing slL-6 and slL-6R results in the formation of a slL-6/slL-6R complex (ZAR067) that is able to bind gp130.

Figure 7 shows that the A2 parent SMIP can bind to the human "native" IL6/SIL6R complex (ZAR067).

Figure 8 shows that the A2M1 SMIP binds to hyper IL6 (ZAR041 ) about

4-5 times better than the parent A2 SMIP.

Figure 9 shows that the A2M1 SMIP (M1 -3/1 1 ) binds to the "native" IL6/SIL6R complex (ZAR067) about 7.5 times better than the A2 SMIP (A2-3/1 1 and A2-4/09), see Figure 9A, using native IL6xlL6R complex from supernatants from HEK293 cells (9A) and from CHO cells (9B).

Figure 10 shows that A2M1 SMIP binds to the "native" IL6/slL6R complex (ZAR074) better than ATH104 and ATH106, two other affinity matured A2 SMIP variants.

Figures 1 1A and 1 1 B show the proliferation in response to recombinant human IL-6 (A) and human hyper IL-6 (B) of the BAF3 cell lines established as described in Example 3. Figures 12A, 12B and 12C show the proliferation of BAF3 cell lines in response to recombinant, non-mammalian derived human rlL-6 (A), slL-6R (B), and hyper IL-6 (C).

Figure 13 shows the proliferation of BAF3 cell lines in response to mammalian-derived human IL-6/slL-6R complex (ZAR067).

Figure 14 shows that the A2 and A2M1 SMIPs neutralized the human slL6xR induced proliferation of BAF3/hgp130 cells. In particular, the A2M1 SMIP was effective with both mammalian-derived (ZAR067) (14B) and non- mammalian derived (14A) slL6xR complexes.

Figure 15 shows that BAF3/hgp130 cell lines proliferated in response to the native human plasma-derived IL-6/slL-6R complex. Cells were incubated for 72 hours in presence of various dilutions of normal human plasma (closed circle) or human LPS plasma (closed square). Proliferation of BAF3/hgp130 cells was measured by ³H-Thymidine incorporation assay. The results are expressed as mean of cpm ± SD of duplicates.

Figure 16 shows the neutralization of native human-derived IL-6/slL-6R complex-induced proliferation of BAF3/hgp130 cells by recombinant human gp130-Fc fusion protein (rhgp130-Fc) and an anti-hlL-6 antibody (MQ2-13A5). The cells were incubated in 96 well plates for 72 h in the presence of human LPS plasma and various concentrations of anti-hlL-6 antibody (MQ2-13A5; closed circle), rhgp130-Fc (closed triangle) or hlgG (closed square).

Proliferation of BAF3/hgp130 cells was measured by ³H-Thymidine

incorporation assay. The results are expressed as mean of cpm ± SD of duplicates.

Figure 17 shows A2 and A2M1 SMIPs' neutralization of native human- derived IL-6/slL-6R complex-induced proliferation of BAF3/hgp130 cells.

BAF3/hgp130 cells were incubated in 96 well plates for 72 h in the presence of human LPS plasma and various concentrations of A2-M1 SMIP (closed circle), A2 SMIP (closed diamond), rhgp130-FC (closed triangle) or hlgG (closed square). Proliferation of BAF3/hgp130 cells was measured by ³H-Thymidine incorporation assay. The results are expressed as mean of cpm ± SD of duplicates.

Figure 18 shows that A2M1 cannot bind IL-6 and binds IL-6R at high concentrations.

Figure 19 is a diagram of the IL-6 signaling process, showing assembly of IL-6/IL6R with gp130 and subsequent dimerization to form the hexamer signaling complex.

Figure 20 is a bar graph summarizing competitive binding experiments showing that the A2 binding domain binds at Site III of the IL6/IL-6R complex.

Figure 21 is a diagram of hyper IL-6 showing site III residues and predicted A2 epitope residues.

Figure 22 shows binding curves for control AH65 binding (A), A2 (B) and A2M1 (C) against hyper IL-6 mutants as summarized in Table 3.

Figure 23 is an amino acid alignment of the VH and VL of the affinity matured A2 mutants. The sequences for the VH and VL for A2, A2M1 ,

ATH072, ATH104 and ATH106 are set forth in SEQ ID NOs:3, 4, 13, 14, 19, 20, 24, 25, 29 and 30, respectively.

Figure 24 is a bar graph showing A2M1 neutralization of STAT3 phosphorylation induced by hyper-IL6 in TF1 cells.

Figure 25 is a bar graph showing A2M1 neutralization of STAT3 phosphorylation induced by HEK-293-derived human slL6xR complex in

BAF3/hgp130 cells.

Figure 26 is a bar graph showing A2M1 neutralization of STAT3 phosphorylation induced by CHO-produced human slL6xR complex in

BAF3/hgp130 cells.

Figure 27 shows A2M1 SMIP effectively blocked a native human- derived IL-6/IL-6R complex induced proliferation of BAF3/hgp130 cells. (A2M1 SMIP (closed circle) or rhgp130-Fc (closed square)). DETAILED DESCRIPTION

The present disclosure relates to polypeptides comprising binding domains that specifically bind to the IL-6/slL-6R complex (slL6xR). In particular, the present disclosure relates to the surprising and unexpected discovery of polypeptides and binding domains (described herein) that bind and inhibit the biological activity of a human native form of slL6xR {e.g., as secreted from appropriately transfected cells or as generated from LPS activated blood cells) better than previously described polypeptides that bind to slL6xR (such as those described in WO2010003101 ), see, e.g., the Examples herein. Thus, polypeptides described herein bind to human native slL6xR and block its biological activity. Further, some polypeptides comprising binding domains as described herein bind to human native slL6xR at least 5 times/fold better than previously described binding domains (such as those described in

WO2010003101 ) and block its biological activity but do not affect or minimally affect IL-6 dependent responses mediated by the membrane IL-6R. Thus, the binding domains described herein block IL-6 trans-signaling and will function to do so in vivo in humans.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents define a term that contradicts the term's definition in the application, the definition that appears in this application controls.

In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components unless otherwise indicated. The use of the alternative {e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used synonymously. In addition, it should be understood that the individual fusion proteins or polypeptides derived from the various combinations of the components {e.g., domains) and

substituents described herein, are disclosed by the present application to the same extent as if each fusion protein was set forth individually. Thus, selection of particular components of individual fusion proteins is specifically

contemplated within the scope of the present disclosure.

"Sequence identity," as used herein, refers to the percentage of amino acid residues in one sequence that are identical with the amino acid residues in another reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The percentage sequence identity values are generated by the NCBI BLAST2.0 software as defined by Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, with the parameters set to default values.

A "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well known in the art {see, e.g., WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71 -77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8). In certain embodiments, a conservative substitution includes a leucine to serine

substitution. As used herein, the term "derivative" refers to a modification of one or more amino acid residues of a peptide by chemical or biological means, either with or without an enzyme, e.g., by glycosylation, alkylation, acylation, ester formation, or amide formation. Generally, a "derivative" differs from an

"analogue" in that a parent polypeptide may be the starting material to generate a "derivative," whereas the parent polypeptide may not necessarily be used as the starting material to generate an "analogue." A derivative may have different chemical, biological or physical properties of the parent polypeptide. For example, a derivative may be more hydrophilic or it may have altered reactivity {e.g., a CDR having a substitution that alters its affinity for a target) as compared to the parent polypeptide.

As used herein, unless otherwise provided, a position of an amino acid residue in a variable region of an immunoglobulin molecule is numbered according to the Kabat numbering convention (Kabat, Sequences of Proteins of Immunological Interest, 5^th ed. Bethesda, MD: Public Health Service, National Institutes of Health (1991 )), and a position of an amino acid residue in a constant region of an immunoglobulin molecule is numbered according to EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94).

A "receptor" is a protein molecule present in the plasma membrane or in the cytoplasm of a cell to which a signal molecule (i.e., a ligand, such as a hormone, a neurotransmitter, a toxin or a cytokine) may attach. The binding of the signal molecule to the receptor results in a conformational change of the receptor, which ordinarily initiates a cellular response. However, some ligands merely block receptors without inducing any response (e.g., antagonists).

Some receptor proteins are peripheral membrane proteins, many hormone and neurotransmitter receptors are transmembrane proteins that embedded in the phospholipid bilayer of cell membranes, and another major class of receptors are intracellular proteins such as those for steroid and intracrine peptide hormone receptors.

The term "biological sample" includes a blood sample, biopsy specimen, tissue explant, organ culture, biological fluid (e.g., serum, urine, CSF, synovial fluid) or any other tissue or cell or other preparation from a subject or a biological source. A subject or biological source may, for example, be a human or non-human animal, a primary cell culture or culture adapted cell line including genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, somatic cell hybrid cell lines, immortalized or immortalizable cell lines, differentiated or

differentiate cell lines, transformed cell lines, or the like. In further

embodiments of this disclosure, a subject or biological source may be suspected of having or being at risk for having a disease, disorder or condition, including a malignant disease, disorder or condition or a B cell disorder. In certain embodiments, a subject or biological source may be suspected of having or being at risk for having a hyperproliferative, inflammatory, or autoimmune disease, and in certain other embodiments of this disclosure the subject or biological source may be known to be free of a risk or presence of such disease, disorder, or condition.

"Treatment," "treating" or "ameliorating" refers to either a therapeutic treatment or prophylactic/preventative treatment. A treatment is therapeutic if at least one symptom of disease in an individual receiving treatment improves or a treatment may delay worsening of a progressive disease in an individual, or prevent onset of additional associated diseases and/or symptoms.

A "therapeutically effective amount (or dose)" or "effective amount (or dose)" of a specific binding molecule or compound refers to that amount of the compound sufficient to result in amelioration or prevention of one or more symptoms of the disease being treated in a statistically significant manner. When referring to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously (in the same formulation or concurrently in separate formulations). The term "pharmaceutically acceptable" refers to molecular entities and compositions that typically do not produce allergic or other serious adverse reactions when administered using routes well known in the art.

A "patient in need" refers to a patient at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a binding polypeptide or a composition thereof provided herein.

Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein. Antibodies are known to have variable regions, a hinge region, and constant domains. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

For example, the terms "VL" and "VH" refer to the variable binding region from an antibody light and heavy chain, respectively. The variable binding regions are made up of discrete, well-defined sub-regions known as

"complementarity determining regions" (CDRs) and "framework regions" (FRs).The term "CL" refers to an "immunoglobulin light chain constant region" or a "light chain constant region," i.e., a constant region from an antibody light chain. The term "CH" refers to an "immunoglobulin heavy chain constant region" or a "heavy chain constant region," which is further divisible, depending on the antibody isotype into CH1 , CH2, and CH3 (IgA, IgD, IgG), or CH1 , CH2, CH3, and CH4 domains (IgE, IgM). A "Fab" (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CH1 of the heavy chain linked to the light chain via an inter-chain disulfide bond.

An "altered immunoglobulin region" or "altered immunoglobulin domain" refers to an immunoglobulin region with a sequence identity to a wild type immunoglobulin region or domain {e.g., a wild type VL, VH, hinge, CL, CH1 , CH2, CH3, or CH4) of at least about 75% (e.g., about 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%). For example, an "altered immunoglobulin CH1 region" or "altered CH1 region" refers to a CH1 region with a sequence identity to a wild type immunoglobulin CH1 region (e.g., a human CH1 ) of at least about 75% (e.g., about 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%). Similarly, an "altered immunoglobulin CH2 domain" or "altered CH2 domain" refers to a CH2 domain with a sequence identity to a wild type immunoglobulin CH1 region (e.g., a human CH2) of at least about 75% (e.g., about 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).

A "wild type immunoglobulin region" or "wild type immunoglobulin domain" refers to a naturally occurring immunoglobulin region or domain (e.g., a naturally occurring VL, VH, hinge, CL, CH1 , CH2, CH3, or CH4) from various immunoglobulin classes or subclasses (including, for example, lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, and IgM) and from various species (including, for example, human, sheep, mouse, rat, and other mammals). Exemplary wild type human CH1 regions are set forth in SEQ ID NOS:82-90, wild type human CK region in SEQ ID NO:91 , wild type human CA regions in SEQ ID NO:92-95, wild type human CH2 domains in SEQ ID NOS:96-104, wild type human CH3 domains in SEQ ID NOS:105-1 13, and wild type human CH4 domains in SEQ ID NO:1 14-1 15.

The invention also includes altered binding molecules, altered binding domains and altered immunoglobulin domains, such as altered VH, VL, scFv, Fc, CH2 or CH3 domains. In certain embodiments, an altered domain (e.g., immunoglobulin domain, VH chain, VL chain, scFv, etc.), only contains conservative amino acid substitutions, e.g., of a wild type immunoglobulin domain. In certain other embodiments, an altered domain only contains non- conservative amino acid substitutions. In yet other embodiments, an altered domain contains both conservative and non-conservative amino acid

substitutions.

Additional definitions are provided throughout the present disclosure. One embodiment of the present disclosure provides an isolated polypeptide comprising a binding domain that specifically binds a SIL6/IL6R (slL6xR) complex, wherein the binding domain and/or polypeptide (a) binds to the IL6xR complex with a higher affinity than either IL6 or IL6Ra alone, or binds to the IL6xR complex alone and to IL6Ra alone with a higher affinity than IL6 alone; (b) competes with membrane gp130 for binding to the slL6xR complex, wherein the binding domain preferentially inhibits IL6 trans-signaling over IL6 cis-signaling and the polypeptide is not a gp130; and (c) inhibits the biological activity of a human native slL6xR complex. In this regard, the biological activity may comprise cell proliferation and/or STAT3 phosphorylation induced by a human native slL6xR complex. Cell proliferation may comprise an appropriate cell line known to the skilled person to respond to human native slL6xR, such as a cell line modified to express gp130 (see e.g., BAF3/hgp130 cells (modified BAF3 cells) as described in the examples herein). STAT3 phosphorylation can similarly be assayed using techniques known in the art and cell lines responsive to human native slL6xR. In one embodiment, the inhibition of the biological activity of a human native slL6xR complex by the polypeptides comprising binding domains as described herein is statistically significant as compared to an isolated polypeptide comprising a binding domain comprising a VH and a VL having the amino acid sequences as set forth in SEQ ID NOs: 3 and 4, respectively, or as compared to a polypeptide with an amino acid sequence consisting of SEQ ID NO:2.

Some embodiments of the disclosure provide an isolated polypeptide that binds to slL6xR wherein the isolated polypeptide comprises from amino- terminus to carboxy-terminus: (a) a binding domain, wherein the binding domain binds to human native slL6xR about at least 5-fold better, e.g., in an ELISA, than a polypeptide comprising a binding domain comprising the VH and VL amino acid sequences as set forth in SEQ ID NOs:3 and 4, respectively, or better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2, and inhibits the biological activity of the human native slL6xR complex; (b) a hinge region; (c) an immunoglobulin heavy chain CH2 constant region polypeptide, and (d) an immunoglobulin heavy chain CH3 constant region polypeptide. In one embodiment of the isolated polypeptides described herein, the binding domain comprises: (a) a VL region comprising a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ; or (b) a VH region comprising a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or (c) a VL of (a) and a VH of (b).

Another aspect of the present disclosure provides a fusion polypeptide comprising: an Fc region constant domain; a hinge region disposed C-terminal to the Fc region constant domain; and a binding domain, wherein the binding domain binds to human native slL6xR at least about 5-fold better, e.g., in an ELISA, than an isolated polypeptide comprising a binding domain comprising the VH and VL amino acid sequences as set forth in SEQ ID NOs:3 and 4, respectively, or than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2 and the polypeptide inhibits the biological activity of the human native slL6xR complex.

In some embodiments, an isolated polypeptide of the disclosure binds to human native slL6xR at least about 5-fold better, e.g., in an ELISA, than a polypeptide comprising a binding domain that binds to hyper-IL6 at a site III epitope. In another embodiment, the isolated polypeptide binds to human native slL6xR at least about 5-fold better, e.g., in an ELISA, than a polypeptide comprising a binding domain comprising the VH and VL amino acid sequences as set forth in SEQ ID NOs:3 and 4, respectively, or than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2.

As used herein, "IL6xR complex" or "IL6xR" refers to a complex of an IL6 with an IL6 receptor, wherein the IL6 receptor (also known as, for example, IL6Ra, IL6RA, IL6R1 , and CD126) is either a membrane protein (referred to herein as mlL6R or mlL6Ra) or a soluble form (referred to herein as slL6R or slL6Ra). The term "IL6R" encompasses both mlL6R and slL6R. In one embodiment, IL6xR comprises a complex of IL6 and mlL6R and may be referred to as mlL6xR. In another embodiment, IL6xR comprises a complex of IL6 and slL6R and is referred to as slL6xR. In certain embodiments, the IL6xR complex is held together via one or more covalent bonds. For example, the carboxy terminus of an IL6R can be fused to the amino-terminus of an IL6 via a peptide linker, which is known in the art as a hyper-IL6 (see, e.g., Fischer et al. (1997) Nat. Biotechnol. 15:142). A hyper-IL6 linker can be comprised of a cross-linking compound, a one to 50 amino acid sequence, or a combination thereof. A hyper-IL6 may further include a dimerization domain, such as an immunoglobulin Fc region constant domain or other immunoglobulin constant domain [e.g., CH1 or CL). One example of a hyper-IL6 is a polypeptide with the amino acid sequence of SEQ ID NO:749). In certain embodiments, the IL6xR complex is held together via non-covalent interactions, such as by hydrogen bonding, electrostatic interactions, Van der Waal's forces, salt bridges, hydrophobic interactions, or the like, or any combination thereof. For example, an IL6 and IL6R or slL6R can naturally associate non-covalently [e.g., as found in nature, or as synthetic or recombinant proteins; see also the examples herein describing in vitro generation of "native" slL6xR from

transfected cell lines and also from human plasma) or each can be fused to a domain that promotes multimerization, such as an immunoglobulin Fc domain, to further enhance complex stability.

A "binding domain" or "binding region," as used herein, refers to a protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a target {e.g., slL6xR). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding domains include single chain antibody variable regions {e.g., domain antibodies, sFv, scFv, Fab, Fab', F(ab')2, Fv), receptor ectodomains {e.g., TNFR), or ligands {e.g., cytokines, chemokines). A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, including Western blot, ELISA, and Biacore analysis.

A binding domain (or a polypeptide comprising a binding domain)

"specifically binds" a target if it binds the target with an affinity or Ka {i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10⁵ M^~1 , while not significantly binding other components present in a test sample. Binding domains (or polypeptides comprising binding domains) may be classified as "high affinity" binding domains and "low affinity" binding domains. "High affinity" binding domains (or polypeptides comprising binding domains) refer to those binding domains with a K_a of at least 10⁷ M^"1, at least 10⁸ M^"1, at least 10⁹ M^"1 , at least 10¹⁰ M^"1, at least 10¹¹ M^"1 , at least 10¹² M^"1 , or at least 10¹³ M^"1. "Low affinity" binding domains (or polypeptides comprising binding domains) refer to those binding domains with a K_a of up to 10⁷ M^"1, up to 10⁶ M^"1 , up to 10⁵ M^~1. Alternatively, affinity may be defined as an equilibrium dissociation constant (K_d) of a particular binding interaction with units of M (e.g., 10^"5 M to 10^"13 M). Affinities of binding domain polypeptides and fusion proteins according to the present disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51 :660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent). In some embodiments, a polypeptide and/or binding domain of the disclosure specifically binds a target, such as slL6xR complex or TNF-a.

One aspect of the present disclosure provides binding polypeptides which bind to IL6xR or slL6xR. In particular, the binding polypeptides of the present disclosure inhibit IL-6 trans-signaling.

As used herein, "gp130" refers to a signal transduction protein that binds to an IL6xR complex. The gp130 protein can be in a membrane (mgp130), soluble (sgpl 30), or any other functional form thereof. Exemplary gp130 proteins have a sequence as set forth in GenBank Accession No. NP_002175.2 or any soluble or derivative form thereof (see, e.g., Narazaki et al. (1993) Blood 82:1 120 or Diamant et al. (1997) FEBS Lett. 412:379). By way of illustration and not wishing to be bound by theory, an mgp130 protein can bind to either an IL6/mlLR or an IL6/slL6R complex, whereas a sgpl 30 primarily binds with an IL6/slLR complex (see Scheller et al. (2006) Scand. J. Immunol. 63:321 ). Thus, certain embodiments of binding domains or binding polypeptides comprising such binding domains of the instant disclosure can inhibit slL6xR complex trans-signaling by binding with higher affinity to slL6xR than to either IL6 or IL6Ra alone and/or by competing with slL6xR complex binding to mgp130. In some embodiments, a binding domain of the instant disclosure "competes" with gp130 binding to a slL6xR when a binding domain or binding polypeptide thereof prevents gp130 from binding a slL6xR and the binding domain binds slL6xR with equal or higher affinity as compared to the binding of gp130 with slL6xR.

In certain embodiments, a binding polypeptide of this disclosure comprises a binding domain that (a) binds to a slL6xR complex with an affinity at least 2-fold, 10-fold, 25-fold, 50-fold, 75-fold to 100-fold, 100-fold to 1000-fold higher than for either IL6 or IL6Ra alone and/or (b) competes with membrane gp130 for binding to slL6xR complex. In further embodiments, a binding domain of this disclosure that binds to a slL6xR complex with an affinity at least 2-fold, 10-fold, 25-fold, 50-fold, 75-fold to 100-fold, 100-fold to 1000-fold higher than for either IL6 or IL6Ra alone may also (i) more significantly or preferentially inhibit IL6 trans-signaling over IL6 cis-signaling, (ii) not inhibit signaling of gp130 cytokine family members other than IL6, (iii) preferentially inhibit IL6 trans-signaling over IL6 cis-signaling and not detectably inhibit signaling of gp130 family cytokines other than IL6, (iv) may have two or more of these properties, or (v) may have all of these properties.

Some polypeptides and binding domains of the invention bind to hyper IL6 {e.g., a polypeptide with an amino acid sequence consisting of SEQ ID NO:749) with a binding affinity of kD 431 pM

In certain embodiments, a slL6xR binding domain of this disclosure binds to a slL6xR complex with an affinity at least 2-fold to 1000-fold higher than for either IL6 or IL6Ra alone and more significantly or preferentially inhibits IL6 trans-signaling over IL6 cis-signaling. To "preferentially inhibit IL6 trans- signaling over IL6 cis-signaling" refers to altering trans-signaling to an extent that slL6xR activity is measurably decreased while the decrease in IL6 cis-signaling is not substantially altered (i.e., meaning inhibition is minimal, non-existent, or not measurable). For example, a biomarker for slL6xR activity {e.g., acute phase expression of antichymotrypsin (ACT) in HepG2 cells or IL6- induced proliferation of TF-1 human erythroleukemic cells; IL-6 induced STAT3 phosphorylation in TF-1 cells) can be measured to detect trans-signaling inhibition. A representative assay is described by Jostock et al. (Eur. J.

Biochem., 2001 ) - briefly, HepG2 cells can be stimulated to overexpress ACT in the presence of slL6xR (trans-signaling) or IL6 (cis-signaling), but adding sgp130 will inhibit the overexpression of ACT induced by slL6xR while not substantially affecting IL6 induced expression. Similarly, a polypeptide binding domain of this disclosure that preferentially inhibits IL6 trans-signaling over IL6 cis-signaling will inhibit the overexpression of ACT induced by slL6xR (i.e., inhibit trans-signaling) while not substantially affecting IL6 induced expression (e.g., not measurably decrease cis-signaling). This and other assays known in the art can be used to measure preferential inhibition of IL6 trans-signaling over IL6 cis-signaling (see, e.g., the BAF3/hgp130 proliferation assay as described in Example 3, STAT3 phosphorylation assays as described herein and known in the art, and other assays described in Sporri et al. (1999) Int. Immunol.

1 1 :1053; Mihara et al. (1995) Br. J. Rheum. 34:321 ; Chen et al. (2004) Immun. 20:59).

In certain embodiments, a slL6xR binding domain described herein binds to a human native slL6xR complex with an affinity or better binding of at least 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21 -fold, 25-fold, 30-fold, 35-fold, 40-fold, 45- fold, 50-fold, 60-fold, 65-fold, 70-fold or higher, such as 1000-fold higher or 5-25 fold, 6-25 fold, 7-25 fold, 8-25 fold, 9-25 fold, 10-25 fold, 5-21 fold, 5-20 fold, 5- 19 fold, 5-18 fold, 5-17 fold, 5-16 fold, 6-20 fold, 7-20 fold, 8-20 fold, 8-19 fold, 8-18 fold, 8-17 fold, 5-10 fold, 10-15 fold, 15-20 fold, 20-25 fold, 25-30 fold, 30- 35 fold, or 35-40 fold than 1 ) a polypeptide with an amino acid sequence consisting of SEQ ID NO:2; and/or 2) a binding polypeptide comprising an anti- IL6xR binding domain having the VH and VL amino acid sequences as set forth in SEQ ID NOs:3 and 4, respectively. In some embodiments, binding or affinity is measured using an ELISA or a BiaCore instrument.

In yet a further embodiment, a polypeptide comprising a slL6xR binding domain described herein inhibits in a statistically significant manner, the biological activity of a human native slL6xR complex as compared to 1 ) a polypeptide with an amino acid sequence consisting of SEQ ID NO:2; and/or 2) a binding polypeptide comprising an anti-IL6xR binding domain having the VH and VL amino acid sequences as set forth in SEQ ID NOs:3 and 4, respectively. In this regard, biological activity may comprise cell proliferation or STAT3 phosphorylation induced by human native IL6xlLR complex. In some embodiments, a polypeptide of the invention may inhibit biological activity of a human native IL6xR complex to a level of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 1 -75%, 5-75%, 10-75%, 15-75%, 20-75%, 30-75%, 40-75%, 50-75%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5- 25%, 5-20%, 20-60%, 30-60%, 40-60%, 40-50% or 30-50% of that obtained with 1 ) a polypeptide with an amino acid sequence consisting of SEQ ID NO:2; and/or 2) a binding polypeptide comprising an anti-IL6xR binding domain having the VH and VL amino acid sequences as set forth in SEQ ID NOs:3 and 4, respectively.

A human native slL6xR complex can be prepared using methodologies known in the art. In one embodiment, a human native slL6xR complex can be prepared recombinantly by expressing human IL6 and human slL6R in an appropriate cell and harvesting the complex from supernatants (see e.g., the Examples described herein). In another embodiment, a human native slL6xR complex can be induced from human plasma by exposure to LPS and harvested/isolated therefrom (see also the Examples herein).

Various binding assays known in the art can be used to assess binding of binding domains as described herein to slL6xR complexes and binding generally of a binding domain to its target. Such assays include ELISA, BiaCore binding assays, competitive binding assays, and the like, using appropriate controls. In another embodiment, a polypeptide comprising a slL6xR binding domain of the present disclosure binds to a mutated Site III epitope of the IL-6 signaling complex, wherein the mutated Site III epitope comprises at least mutations at one or two positions selected from F134, 1170 and R132 of the IL6R amino acid sequence present in the mature form (i.e., without the leader peptide) of the hyper-IL6 fusion protein as set forth in SEQ ID NO:749. In this regard, as noted elsewhere, IL-6 assembles with IL-6R and GP130 to form a trimeric complex (see Figure 19); Site I and II drive complex formation. The IL- 6 signaling complex is a hexamer comprised of two of the IL-6/mlL-6R/gp130 trimeric complexes (see Figure 19); Site III drives dimerization of the trimeric complexes to form the hexameric signaling complex, and subsequent signaling. Mutation of F134, 1170 and R132 positions of the IL6R abrogates binding of the polypeptides comprising the binding domain having the VH and VL as set forth in SEQ ID NOs:3 and 4. (Figure 22) However, in some embodiments of the invention, a slL6xR binding domain of the present disclosure maintains the ability to bind to such mutants. In one embodiment, the mutations comprise any one or more of the following substitutions: F134G, 1170T, and R132G and a polypeptide comprising a slL6xR binding domain of the present disclosure maintains binding. In some embodiments, however, a polypeptide comprising a slL6xR binding domain of the present disclosure may bind site III mutants having other amino acid or amino acid analog substitutions at these positions.

In some embodiments, signaling by gp130 family cytokines other than IL6 is not substantially inhibited by binding domain polypeptides or multi- specific fusion proteins thereof of this disclosure, e.g., signaling by one or more other gp130 family cytokines will be minimally affected or unaffected, such as signaling via leukemia inhibitory factor (LIF), ciliary neurotropic factor (CNTF), neuropoietin (NPN), cardiotropin like cytokine (CLC), oncostatin M (OSM), IL- 1 1 , IL-27, IL-31 , cardiotrophin-1 (CT-1 ), or any combination thereof.

It will be appreciated by those skilled in the art that a longer in vivo half- life is generally desirable and in some embodiments a binding domain of this disclosure is on the order of days or weeks. While in some cases the binding domain concentration in vivo may be low, the target may be plentiful as both IL6 and slL6 production can be quite elevated in disease states (see, e.g., Lu et al. (1993) Cytokine 5:578). Thus, in certain embodiments, a binding domain of this disclosure has a k₀FF of about 10^"5/sec (e.g., about a day) or less. In certain embodiments, the k₀FF can range from about 10^"Vsec, about 10^"2/sec, about 10^{" 3}/sec, about 10^"4/sec, about 10^"5/sec, about 10^"6/sec, about 10^"7/sec, about 10^{" 8}/sec, about 10^"9/sec, about 10^"10/sec, or less.

Synthetic IL6xR complexes are known in the art and may be used herein for binding assays to test the slL6xR binding domains of the present disclosure. In certain embodiments, synthetic IL6xR may comprises a structure of N-

IL6Ra(frag)-L1 -IL6(frag)-L2-ID-C, wherein N is the amino-terminus and C is the carboxy-terminus, IL6Ra(frag) is a fragment of full length IL6Ra, IL6(frag) is a fragment of IL6, L1 and L2 are linkers, and ID is an intervening or dimerization domain, such as an immunoglobulin Fc domain.

In other embodiments, an IL6xR (such as a form of hyper IL6) which may be used to assess binding of the binding domains specific for IL6xR complex has a structure, from amino-terminus to carboxy-terminus, as follows: (1 ) a central fragment of 212 amino acids from IL6Ra that is missing the first 1 10 amino acids of the full length protein and a carboxy-terminal portion that will depend on the isoform used (see GenBank Accession No. NP_000556.1 , isoform 1 or NP_852004.1 , isoform 2) fused to (2) a linker of G₃S that is in turn fused to (3) a 175 amino acid carboxy-terminal fragment of IL6 (i.e., missing the first 27 amino acids of the full length protein; GenBank Accession No.

NP_000591 .1 ) that is in turn fused to (4) a linker, e.g., that is an lgG2A hinge, e.g., as set forth in SEQ ID NO:1 16, which is fused to a dimerization domain, e.g., comprised of an immunoglobulin G1 (lgG1 ) Fc domain. In certain embodiments, the dimerization domain comprised of an lgG1 Fc domain has one or more of the following amino acids mutated (i.e., have a different amino acid at that position): leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), glutamate at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (EU numbering). For example, any one of these amino acids can be changed to alanine. In a further embodiment, an lgG1 Fc domain has each of L234, L235, G237, E318, K320, and K322 (according to EU numbering) mutated to an alanine (i.e., L234A, L235A, G237A, E318A, K320A, and K322A, respectively).

In one embodiment, an IL6xR complex which may be used to assess binding of the binding domains of this disclosure has an amino acid sequence as set forth in SEQ ID NO:1 17. In certain embodiments, there are provided polypeptides containing a binding domain specific for an IL6xR complex, wherein the IL6xR is a slL6xR and has the amino acid sequence as set forth in SEQ ID NO:1 17.

In other embodiments, polypeptides of the disclosure may contain a binding domain specific for an IL6xR complex that (1 ) has greater or equal affinity for an IL6xR complex than for IL6 or IL6Ra alone, or has greater affinity for IL6R alone or an IL6xR complex than for IL6 alone, (2) competes with membrane gp130 for binding with a slL6xR complex, (3) preferentially inhibits IL6 trans-signaling over IL6 cis-signaling (4) does not inhibit signaling of gp130 family cytokines other than IL6, (5) has greater affinity for human native slL6xR complex than binding polypeptides comprising a binding domain having the VH and VL amino acid sequences set forth in SEQ ID NOs:3 and 4, respectively, or than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2; (6) inhibits the biological activity of a human native slL6xR complex; (7) has any combination thereof of properties (1 ) - (6); or (8) has all of the properties of (1 ) - (6). Other exemplary IL6xR complexes that may be used to assess binding or to identify binding domains of the instant disclosure or used as a reference complex to measure any of the aforementioned binding properties are described, for example, in US Patent Publication Nos. 2007/0172458;

2007/0031376; and US Patent Nos. 7,198,781 ; 5,919,763.

As indicated above, a polypeptide of the present disclosure comprises a binding domain that specifically binds a target [e.g., slL6xR). Binding of a target by the binding domain may block the interaction between the target [e.g., slL6xR) and another molecule (e.g., gp130), and thus interfere, reduce or eliminate certain functions of the target [e.g., signal transduction).

Thus, a binding domain may be any peptide that specifically binds a target of interest [e.g., slL6xR). Sources of binding domains include antibody variable regions from various species (which can be formatted as antibodies, sFvs, scFvs (see, e.g., Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-83, 1988), Fabs, or soluble VH region or domain antibodies), including human, rodent, avian, and ovine. Domain antibodies (dAbs) comprise a variable region of a heavy or light chain of an immunoglobulin (V_H and V_L, respectively) (Holt et al., (2003) Trends Biotechnol. 21 :484-490). Additional sources of binding domains include variable regions of antibodies from other species, such as camelid (from camels, dromedaries, or llamas; Ghahroudi et al. (1997) FEBS Letters 414(3):521 -526; Vincke et al. (2009) Journal of Biological Chemistry (2009) 284:3273-3284; Hamers-Casterman et al. (1993) Nature, 363:446 and Nguyen et al. (1998) J. Mol. Biol., 275:413), nurse sharks (Roux et al. (1998) Proc. Nat'l. Acad. Sci. (USA) 95:1 1804), spotted ratfish (Nguyen et al. (2002) Immunogenetics, 54:39), or lamprey (Herrin et al., (2008) Proc. Nat'l. Acad. Sci. (USA) 105:2040-2045 and Alder et al. (2008) Nature Immunology 9:319-327). These antibodies can apparently form antigen-binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as "heavy chain antibodies") (Jespers et al. (2004) Nature Biotechnology 22:1 161 -1 165; Cortez-Retamozo et al. (2004) Cancer Research 64:2853-2857; Baral et al. (2006) Nature Medicine 12:580-584, and Barthelemy et al. (2008) Journal of Biological Chemistry 283:3639-3654).

An alternative source of binding domains of this disclosure includes sequences that encode random peptide libraries or sequences that encode an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds, such as fibrinogen domains (see, e.g., Weisel et al. (1985) Science 230:1388), Kunitz domains (see, e.g., US Patent No. 6,423,498), ankyrin repeat proteins (Binz et al. (2003) Journal of Molecular Biology 332:489-503 and Binz et al. (2004) Nature Biotechnology 22(5):575-582), fibronectin binding domains (Richards et al. (2003) Journal of Molecular Biology 326:1475-1488; Parker et al. (2005) Protein Engineering Design and Selection 18(9):435-444 and Hackel et al. (2008) Journal of Molecular Biology 381 :1238-1252), cysteine-knot miniproteins (Vita et al. (1995) Proc. Nat'l. Acad. Sci. (USA) 92:6404-6408; Martin et al. (2002) Nature Biotechnology 21 :71 -76 and Huang et al. (2005) Structure 13:755-768), tetratricopeptide repeat domains (Main et al. (2003) Structure 1 1 :497-508 and Cortajarena et al. (2008) ACS Chemical Biology 3:161 -166), leucine-rich repeat domains (Stumpp et al. (2003) Journal of Molecular Biology 332:471 -487), lipocalin domains (see, e.g., WO

2006/095164, Beste et al. (1999) Proc. Nat'l. Acad. Sci. (USA) 96:1898-1903 and Schonfeld et al. (2009) Proc. Nat'l. Acad. Sci. (USA) 106:8198-8203), V-like domains (see, e.g., U.S. Patent Application Publication No. 2007/0065431 ), C- type lectin domains (Zelensky and Gready (2005) FEBS J. 272:6179; Beavil et al. (1992) Proc. Nat'l. Acad. Sci. (USA) 89:753-757 and Sato et al. (2003) Proc. Nat'l. Acad. Sci. (USA) 100:7779-7784), mAb² or Fcab™ (see, e.g., PCT Patent Application Publication Nos. WO 2007/098934; WO 2006/072620), or the like (Nord et al. (1995) Protein Engineering 8(6):601 -608; Nord et al. (1997) Nature Biotechnology 15:772-777; Nord et al. (2001 ) European Journal of Biochemistry 268(15):4269-4277 and Binz et al. (2005) Nature Biotechnology 23:1257-1268).

Binding domains of this disclosure can be generated as described herein or by a variety of methods known in the art (see, e.g., U.S. Patent Nos.

6,291 ,161 and 6,291 ,158). For example, binding domains of this disclosure may be identified by screening a Fab phage library for Fab fragments that specifically bind to a target of interest (see Hoet et al. (2005) Nature Biotechnol. 23:344). Additionally, traditional strategies for hybridoma development using a target of interest as an immunogen in convenient systems (e.g., mice, HUMAb MOUSE®, TC MOUSE™, KM-MOUSE^®, llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains of this disclosure.

In some embodiments, a binding domain is a single chain Fv fragment (scFv) that comprises V_H and V_L regions specific for a target of interest. In certain embodiments, the V_H and VL regions are human. Exemplary V_L and V_H regions include the V_L and V_H regions of the A2M1 binding domain as described herein, and other related affinity matured mutants derived from the A2 binding domain. Amino acid sequences of exemplary VH regions are set forth in SEQ ID NOs:13, 19, 24, and 29. Amino acid sequences of exemplary VL regions are set forth in SEQ ID NOs:14, 20, 25, and 30.

In certain embodiments, a binding domain comprises or is a sequence that is at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (V_L) (e.g., SEQ ID NOS: 14, 20, 25 or 30) or to a heavy chain variable region (V_H) {e.g., SEQ ID NOS:13, 19, 24, 29), or both. In certain embodiments, each CDR comprises no more than one, two, or three substitutions, insertions or deletions, as compared to that from a monoclonal antibody or fragment or derivative thereof that specifically binds to a target of interest {e.g., slL6xR). In further embodiments, a binding domain comprises a CDR1 , CDR2 and CDR3 {e.g., CDR1 , CDR2 and CDR3 from the A2M1 binding domain as described herein) wherein one, two, or three of the CDRs comprise a fragment of a CDR as disclosed herein, such as a fragment of a CDR having 3, 4, 5, 6, 7, 8, or 9 amino acids of a CDR described herein.

In certain embodiments, a binding domain comprises or is a sequence that is a humanized version of a light chain variable region (V_L) {e.g., SEQ ID NOS: 14, 20, 25, or 30) or a heavy chain variable region (V_H) {e.g., SEQ ID NOS:13, 19, 24 or 29), or both.

In certain embodiments, a binding domain V_H region of the present disclosure can be derived from or based on a V_H of a known monoclonal antibody {e.g., AH-65 or CLB-16 anti IL6xR antibodies) or a V_H described herein and contains about one or more {e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more {e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more {e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions {e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH of a known monoclonal antibody. The insertion(s), deletion(s) or substitution(s) may be anywhere in the VH region, including at the amino- or carboxyl-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes and provided a binding domain containing the modified VH region can still specifically bind its target with an affinity similar to or better than the original/unmodified/parental binding domain.

In further embodiments, a VL region in a binding domain of the present disclosure is derived from or based on a VL of a known monoclonal antibody or a VL described herein and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g. , conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL of the known monoclonal antibody. The insertion(s), deletion(s) or substitution(s) may be anywhere in the VL region, including at the amino- or carboxyl-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes and provided a binding domain containing the modified V_L region can still

specifically bind its target with an affinity similar to or better than the

original/unmodified/parental binding domain.

The VH and VL regions may be arranged in either orientation (i.e., from amino-terminus to carboxy terminus, VH-VL or VL-VH) and may optionally be joined by a variable domain linker, e.g. , an amino acid sequence (e.g., having a length of about five to about 35 amino acids) capable of providing a spacer function such that the two sub-binding domains can interact to form a functional binding domain. In certain embodiments, an amino acid sequence that joins the VH and VL regions (also referred to herein as a "variable domain linker") includes those belonging to the (Gly_nSer) family, such as (Gly₃Ser)_n(Gly₄Ser)i ,

(Gly₃Ser)n(Gly₄Ser)_n, or (Gly₄Ser)_n, wherein n is an integer of 1 to 5. In certain embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO:361 ) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:362). In certain embodiments, these (Gly_nSer)-based linkers are used to link the VH and VL regions in a binding domain, but are not used to link a binding domain to any other domain, e.g., a heterodimerization domain or to an Fc region portion.

Exemplary binding domains specific for slL6xR include an A2M1 scFv comprising the VH and VL as set forth in SEQ ID NOs:13 and 14 and the (G₄S)₃ variable domain linker as set forth in SEQ ID NO:33, or humanized versions thereof. Other binding domains specific for slL6xR include an scFv comprising the VH amino acid sequence as set forth in one of SEQ ID NOs:19, 24 or 29 and the VL amino acid sequence as set forth in one of SEQ ID NOs:20, 25 or 30, and al linker such as the (G₄S)3 variable domain linker as set forth in SEQ ID NO:33, or humanized versions thereof. Examples of scFvs are the binding polypeptides as set forth in SEQ ID NOs:12, 28, 23 and 28, encoded by the polynucleotides as set forth in SEQ ID NOs:1 1 , 27, 22 and 27, respectively.

In certain embodiments, an isolated polypeptide comprising a binding domain as described herein is in the form of an antibody or antigen binding fragment thereof, such as F(ab), F(ab')2, Fv, sFv, and scFv. Monoclonal antibodies specific for slL6xR or other target of interest may be prepared, for example, using the techniques well known in the art, such as the techniques of Kohler and Milstein, Eur. J. Immunol. 6:51 1 -519, 1976, and improvements thereto; Wayner EA, Hoffstrom BG. 2007. Methods Enzymol 426: 1 17-153; and Lane RD. 1985. J Immunol Methods 81 : 223-228.

These methods include the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, such as one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. One selection technique uses HAT (hypoxanthine, aminopterin, and thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas producing antibodies having high reactivity and/or specificity are generally selected.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the

peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood.

Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.

The present disclosure provides fusion proteins and polypeptides comprising binding domains, in particular, binding domains that specifically bind slL6xR. The fusion proteins comprising the binding domains as described herein in certain embodiments comprise any of a variety of other

components/domains such as Fc region domains, linkers, hinges,

dimerization/heterodimerization domains, junctional amino acids, tags, a heterologous binding domain etc.

As indicated herein, the polypeptides comprising binding domains as described herein may comprise an Fc region constant domain portion (also referred to as an Fc region portion or Fc region constant domain). The inclusion of an Fc region portion may slow clearance of the binding proteins from circulation after administration to a subject. By mutations or other alterations, the Fc region portion further enables modulation of effector functions of the binding polypeptide, or dimers or heterodimers thereof, {e.g., ADCC, ADCP, CDC, complement fixation and binding to Fc receptors), which can either be increased or decreased depending on the disease being treated, as known in the art and described herein. In certain embodiments, an Fc region portion of binding polypeptides of the present disclosure will be capable of mediating one or more of these effector functions. In some embodiments, an Fc region portion of binding polypeptides of the present disclosure will have minimal or no capability of mediating one or more of these effector functions As used herein, "an Fc region constant domain portion" or "Fc region portion" refers to the heavy chain constant region segment of the Fc fragment (the "fragment crystallizable" region or Fc region) from an antibody, which can include one or more constant domains, such as CH2, CH3, CH4, or any combination thereof. In certain embodiments, an Fc region constant domain comprises a domain derived from an immunoglobulin CH2 domain, and optionally a domain derived from an immunoglobulin CH3 domain, but does not contain a domain or region derived from, or corresponding to, an

immunoglobulin CH1 domain. In certain embodiments, an Fc region portion includes the CH2 and CH3 domains of an IgG, IgA, or IgD antibody and any combination thereof, or the CH3 and CH4 domains of an IgM or IgE antibody and any combination thereof. In one embodiment, the CH2CH3 or the

CH3CH4 structures are from the same antibody isotype, such as IgG, IgA, IgD, IgE, or IgM. By way of background, the Fc region is responsible for the effector functions of an immunoglobulin, such as ADCC (antibody-dependent cell- mediated cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors {e.g., CD16, CD32, FcRn), greater half-life in vivo relative to a polypeptide lacking an Fc region, protein A binding, and perhaps even placental transfer (see Capon et ai, Nature, 337:525 (1989)). In certain embodiments, an Fc region portion found in binding polypeptide heterodimers of the present disclosure will be capable of mediating one or more of these effector functions.

An Fc region portion present in single chain binding polypeptides may comprise a CH2 domain, a CH3 domain, a CH4 domain or any combination thereof. For example, an Fc region portion may comprise a CH2 domain, a CH3 domain, both CH2 and CH3 domains, both CH3 and CH4 domains, two CH3 domains, a CH4 domain, or two CH4 domains. In some embodiments, a binding molecule of the disclosure comprises an Fc region comprising amino acids 250-480 of SEQ ID NO:12.

A CH2 domain that may form an Fc region portion of a binding

polypeptide of the present disclosure may be a wild type immunoglobulin CH2 domain or an altered immunoglobulin CH2 domain thereof from certain immunoglobulin classes or subclasses (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, or IgD) and from various species (including human, mouse, rat, and other mammals).

In certain embodiments, a CH2 domain is a wild type human

immunoglobulin CH2 domain, such as wild type CH2 domains of human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, or IgD, as set forth in SEQ ID NOS:96, 101 -103 and 97-99, respectively. In certain embodiments, the CH2 domain is a wild type human lgG1 CH2 domain as set forth in SEQ ID NO:96.

In certain embodiments, a CH2 domain is an altered immunoglobulin CH2 region [e.g., an altered human lgG1 CH2 domain) that comprises an amino acid substitution at the asparagine of position 297 [e.g., asparagine to alanine). Such an amino acid substitution reduces or eliminates glycosylation at this site and abrogates efficient Fc binding to FcyR and C1 q. The sequence of an altered human lgG1 CH2 domain with an Asn to Ala substitution at position 297 is set forth in SEQ ID NO:424.

In certain embodiments, a CH2 domain is an altered immunoglobulin CH2 region {e.g., an altered human lgG1 CH2 domain) that comprises at least one substitution or deletion at positions 234 to 238. For example, an

immunoglobulin CH2 region can comprise a substitution at position 234, 235, 236, 237 or 238, positions 234 and 235, positions 234 and 236, positions 234 and 237, positions 234 and 238, positions 234-236, positions 234, 235 and 237, positions 234, 236 and 238, positions 234, 235, 237, and 238, positions 236- 238, or any other combination of two, three, four, or five amino acids at positions 234-238. In addition or alternatively, an altered CH2 region may comprise one or more {e.g., two, three, four or five) amino acid deletions at positions 234-238, for instance, a deletion at one of position 236 or position 237 while the other position is substituted. The above-noted mutation(s) decrease or eliminate the antibody-dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a binding polypeptide that comprises the altered CH2 domain. In certain embodiments, the amino acid residues at one or more of positions 234-238 has been replaced with one or more alanine residues. In further embodiments, only one of the amino acid residues at positions 234-238 have been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid {e.g., alanine or serine). In some embodiments, positions 234-237 are replaced three alanines, three serines, one alanine and two serine or two alanines and one serine

In certain other embodiments, a CH2 domain is an altered

immunoglobulin CH2 region {e.g., an altered human lgG1 CH2 domain) that comprises one or more amino acid substitutions at positions 253, 310, 318, 320, 322, and 331 . For example, an immunoglobulin CH2 region can comprise a substitution at position 253, 310, 318, 320, 322, or 331 , positions 318 and 320, positions 318 and 322, positions 318, 320 and 322, or any other combination of two, three, four, five or six amino acids at positions 253, 310, 318, 320, 322, and 331 . The above-noted mutation(s) decrease or eliminate the complement-dependent cytotoxicity (CDC) of a binding polypeptide that comprises the altered CH2 domain.

In certain other embodiments, in addition to the amino acid substitution at position 297, an altered CH2 region {e.g., an altered human lgG1 CH2 domain) can further comprise one or more {e.g., two, three, four, or five) additional substitutions at positions 234-238. For example, an immunoglobulin CH2 region can comprise a substitution at positions 234 and 297, positions 234, 235, and 297, positions 234, 236 and 297, positions 234-236 and 297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or any combination of two, three, four, or five amino acids at positions 234-238 in addition to position 297. In addition or alternatively, an altered CH2 region may comprise one or more (e.g., two, three, four or five) amino acid deletions at positions 234-238, such as at position 236 or position 237. The additional mutation(s) decreases or eliminates the antibody-dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a binding polypeptide that comprises the altered CH2 domain. In certain embodiments, the amino acid residues at one or more of positions 234-238 have been replaced with one or more alanine residues. In further embodiments, only one of the amino acid residues at positions 234-238 has been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid {e.g., alanine or serine).

In certain embodiments, in addition to one or more {e.g., 2, 3, 4, or 5) amino acid substitutions at positions 234-238, a mutated CH2 region {e.g., an altered human lgG1 CH2 domain) in a polypeptide of the present disclosure may contain one or more {e.g., 2, 3, 4, 5, or 6) additional amino acid

substitutions {e.g., substituted with alanine) at one or more positions involved in complement fixation {e.g., at positions I253, H310, E318, K320, K322, or P331 ). Examples of mutated immunoglobulin CH2 regions include human lgG1 , lgG2, lgG4 and mouse lgG2a CH2 regions with alanine substitutions at positions 234, 235, 237 (if present), 318, 320 and 322. An exemplary mutated

immunoglobulin CH2 region is mouse IGHG2c CH2 region with alanine substitutions at L234, L235, G237, E318, K320, and K322 (SEQ ID NO:425).

In still further embodiments, in addition to the amino acid substitution at position 297 and the additional deletion(s) or substitution(s) at positions 234- 238, an altered CH2 region {e.g., an altered human lgG1 CH2 domain) can further comprise one or more {e.g., two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331 . For example, an immunoglobulin CH2 region can comprise a (1 ) substitution at position 297, (2) one or more substitutions or deletions or a combination thereof at positions 234-238, and one or more {e.g., 2, 3, 4, 5, or 6) amino acid substitutions at positions I253, H310, E318, K320, K322, and P331 , such as one, two, three substitutions at positions E318, K320 and K322. In one embodiment, the amino acids at the above-noted positions are substituted by alanine or serine.

In certain embodiments, an immunoglobulin CH2 region polypeptide comprises: (i) an amino acid substitution at the asparagines of position 297 and one amino acid substitution at position 234, 235, 236 or 237; (ii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at two of positions 234-237; (iii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at three of positions 234-237; (iv) an amino acid substitution at the asparagine of position 297, amino acid

substitutions at positions 234, 235 and 237, and an amino acid deletion at position 236; (v) amino acid substitutions at three of positions 234-237 and amino acid substitutions at positions 318, 320 and 322; or (vi) amino acid substitutions at three of positions 234-237, an amino acid deletion at position 236, and amino acid substitutions at positions 318, 320 and 322.

Exemplary altered immunoglobulin CH2 regions with amino acid substitutions at the asparagine of position 297 include: human lgG1 CH2 region with alanine substitutions at L234, L235, G237 and N297 and a deletion at G236 (SEQ ID NO:426), human lgG2 CH2 region with alanine substitutions at V234, G236, and N297 (SEQ ID NO:427), human lgG4 CH2 region with alanine substitutions at F234, L235, G237 and N297 and a deletion of G236 (SEQ ID NO:428), human lgG4 CH2 region with alanine substitutions at F234 and N297 (SEQ ID NO:429), human lgG4 CH2 region with alanine substitutions at L235 and N297 (SEQ ID NO:430), human lgG4 CH2 region with alanine substitutions at G236 and N297 (SEQ ID NO:431 ), and human lgG4 CH2 region with alanine substitutions at G237 and N297 (SEQ ID NO:432).

In certain embodiments, in addition to the amino acid substitutions described above, an altered CH2 region {e.g., an altered human lgG1 CH2 domain) may contain one or more additional amino acid substitutions at one or more positions other than the above-noted positions. Such amino acid substitutions may be conservative or non-conservative amino acid substitutions. For example, in certain embodiments, P233 may be changed to E233 in an altered lgG2 CH2 region (see, e.g., SEQ ID NO:427). In addition or

alternatively, in certain embodiments, the altered CH2 region may contain one or more amino acid insertions, deletions, or both. The insertion(s), deletion(s) or substitution(s) may anywhere in an immunoglobulin CH2 region, such as at the N- or C-terminus of a wild type immunoglobulin CH2 region resulting from linking the CH2 region with another region [e.g., a binding domain or a heterodimerization domain) via a hinge.

In certain embodiments, an altered CH2 region in a binding polypeptide of the present disclosure comprises or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a wild type immunoglobulin CH2 region, such as the CH2 region of wild type human lgG1 , lgG2, or lgG4, or mouse lgG2a (e.g., IGHG2c).

An altered immunoglobulin CH2 region in a binding polypeptide of the present disclosure may be derived from a CH2 region of various

immunoglobulin isotypes, such as lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, and IgD, from various species (including human, mouse, rat, and other mammals). In certain embodiments, an altered immunoglobulin CH2 region in a fusion protein of the present disclosure may be derived from a CH2 region of human lgG1 , lgG2 or lgG4, or mouse lgG2a (e.g., IGHG2c), whose sequences are set forth in SEQ ID NOS:96, 101 , 103 and 433.

In certain embodiments, an altered CH2 domain is a human lgG1 CH2 domain with alanine substitutions at positions 235, 318, 320, and 322 (i.e., a human lgG1 CH2 domain with L235A, E318A, K320A and K322A substitutions) (SEQ ID NO:434), and optionally an N297 mutation (e.g., to alanine). In certain other embodiments, an altered CH2 domain is a human lgG1 CH2 domain with alanine substitutions at positions 234, 235, 237, 318, 320 and 322 (i.e., a human lgG1 CH2 domain with L234A, L235A, G237A, E318A, K320A and K322A substitutions) (SEQ ID NO:435), and optionally an N297 mutation (e.g., to alanine). In certain embodiments, an altered CH2 domain is an altered human lgG1 CH2 domain with mutations known in the art that enhance immunological activities such as ADCC, ADCP, CDC, complement fixation, Fc receptor binding, or any combination thereof.

The CH3 domain that may form an Fc region portion of a binding polypeptide of the present disclosure may be a wild type immunoglobulin CH3 domain or an altered immunoglobulin CH3 domain thereof from certain immunoglobulin classes or subclasses (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, IgM) of various species (including human, mouse, rat, and other mammals). In certain embodiments, a CH3 domain is a wild type human immunoglobulin CH3 domain, such as wild type CH3 domains of human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM as set forth in SEQ ID NOS:436- 445, respectively. In certain embodiments, the CH3 domain is a wild type human lgG1 CH3 domain as set forth in SEQ ID NO:436. In certain

embodiments, a CH3 domain is an altered human immunoglobulin CH3 domain, such as an altered CH3 domain based on or derived from a wild-type CH3 domain of human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM antibodies. For example, an altered CH3 domain may be a human lgG1 CH3 domain with one or two mutations at positions H433 and N434 (positions are numbered according to EU numbering). The mutations in such positions may be involved in complement fixation. In certain other embodiments, an altered CH3 domain may be a human lgG1 CH3 domain but with one or two amino acid substitutions at position F405 or Y407. The amino acids at such positions are involved in interacting with another CH3 domain. In certain embodiments, an altered CH3 domain may be an altered human lgG1 CH3 domain with its last lysine deleted. The sequence of this altered CH3 domain is set forth in SEQ ID NO:446.

In certain embodiments, particularly where a binding polypeptide heterodimer is desired, the polypeptides of the heterodimer comprise a CH3 pair that comprises so called "knobs-into-holes" mutations (see, Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et ai, Protein Engineering 9:617-21 , 1966). More specifically, mutations may be introduced into each of the two CH3 domains so that the steric complementarity required for CH3/CH3 association obligates these two CH3 domains to pair with each other. For example, a CH3 domain in one single chain polypeptide of a binding polypeptide heterodimer may contain a T366W mutation (a "knob" mutation, which substitutes a small amino acid with a larger one), and a CH3 domain in the other single chain polypeptide of the binding polypeptide heterodimer may contain a Y407A mutation (a "hole" mutation, which substitutes a large amino acid with a smaller one). Other exemplary knobs-into-holes mutations include (1 ) a T366Y mutation in one CH3 domain and a Y407T in the other CH3 domain, and (2) a T366W mutation in one CH3 domain and T366S, L368A and Y407V mutations in the other CH3 domain.

The CH4 domain that may form an Fc region portion of a single chain binding polypeptide, which may or may not contain a binding domain, may be a wild type immunoglobulin CH4 domain or an altered immunoglobulin CH4 domain thereof from IgE or IgM molecules. In certain embodiments, the CH4 domain is a wild type human immunoglobulin CH4 domain, such as wild type CH4 domains of human IgE and IgM molecules as set forth in SEQ ID NOS:447 and 448, respectively. In certain embodiments, a CH4 domain is an altered human immunoglobulin CH4 domain, such as an altered CH4 domain based on or derived from a CH4 domain of human IgE or IgM molecules, which have mutations that increase or decrease an immunological activity known to be associated with an IgE or IgM Fc region.

In certain embodiments, an Fc region constant domain portion comprises a combination of CH2, CH3 or CH4 domains (i.e., more than one Fc constant domain selected from CH2, CH3 and CH4). For example, the Fc region portion may comprise CH2 and CH3 domains or CH3 and CH4 domains. In certain other embodiments, the Fc region portion may comprise two CH3 domains and no CH2 or CH4 domains (i.e., only two or more CH3). The multiple constant domains that form an Fc region portion may be based on or derived from the same immunoglobulin molecule, or the same class or subclass immunoglobulin molecules. In certain embodiments, the Fc region portion is an IgG CH2CH3 (e.g., lgG1 CH2CH3, lgG2 CH2CH3, and lgG4 CH2CH3) and in certain embodiments is human [e.g., human lgG1 , lgG2, and lgG4) CH2CH3. For example, in certain embodiments, the Fc region portion comprises (1 ) wild type human lgG1 CH2 and CH3 domains, (2) human lgG1 CH2 with N297A substitution (i.e., CH2(N297A)) and wild type human lgG1 CH3, or (3) human lgG1 CH2(N297A) and an altered human lgG1 CH3 with the last lysine deleted.

Alternatively, the multiple constant domains may be based on or derived from different immunoglobulin molecules, or different classes or subclasses immunoglobulin molecules. For example, in certain embodiments, an Fc region portion comprises both human IgM CH3 domain and human lgG1 CH3 domain. The multiple constant domains that form an Fc region portion may be directly linked together or may be linked to each other via one or more {e.g., 2-8) amino acids.

Exemplary Fc region portions are set forth in SEQ ID NOS:449-460.

With regard to heterodimers as disclosed herein, in certain

embodiments, the Fc region portions of both single chain polypeptides of a binding polypeptide heterodimer are identical to each other. In certain other embodiments, the Fc region portion of one single chain polypeptide of a binding polypeptide heterodimer is different from the Fc region portion of the other single chain binding polypeptide of the heterodimer. For example, one Fc region portion may contain a CH3 domain with a "knob" mutation, whereas the other Fc region portion may contain a CH3 domain with a "hole" mutation.

In certain embodiments, an immunoglobulin Fc region {e.g., CH2, CH3, and/or CH4 regions) may have an altered glycosylation pattern relative to an immunoglobulin reference sequence. For example, any of a variety of genetic techniques may be employed to alter one or more particular amino acid residues that form a glycosylation site (see Co et al. (1993) Mol. Immunol.

30:1361 ; Jacquemon et al. (2006) J. Thromb. Haemost. 4:1047; Schuster et al. (2005) Cancer Res. 65:7934; Warnock et al. (2005) Biotechnol. Bioeng.

92:831 ), such as N297 of the CH2 domain (EU numbering). Alternatively, the host cells producing the binding polypeptides may be engineered to produce an altered glycosylation pattern. One method known in the art, for example, provides altered glycosylation in the form of bisected, non-fucosylated variants that increase ADCC. The variants result from expression in a host cell containing an oligosaccharide-modifying enzyme. Alternatively, the Potelligent technology of BioWa/Kyowa Hakko is contemplated to reduce the fucose content of glycosylated molecules according to this disclosure. In one known method, a CHO host cell for recombinant immunoglobulin production is provided that modifies the glycosylation pattern of the immunoglobulin Fc region, through production of GDP-fucose.

Alternatively, chemical techniques are used to alter the glycosylation pattern of fusion polypeptide of this disclosure. For example, a variety of glycosidase and/or mannosidase inhibitors provide one or more of desired effects of increasing ADCC activity, increasing Fc receptor binding, and altering glycosylation pattern. In certain embodiments, cells expressing fusion polypeptides of the instant disclosure are grown in a culture medium comprising a carbohydrate modifier at a concentration that increases the ADCC of immunoglycoprotein molecules produced by said host cell, wherein said carbohydrate modifier is at a concentration of less than 800 μΜ. In one embodiment, the cells expressing these polypeptides are grown in a culture medium comprising castanospermine or kifunensine, for instance,

castanospermine at a concentration of 100-800 μΜ, such as 100 μΜ, 200 μΜ, 300 μΜ, 400 μΜ, 500 μΜ, 600 μΜ, 700 μΜ, or 800 μΜ. Methods for altering glycosylation with a carbohydrate modifier such as castanospermine are provided in U.S. Patent No. 7846434, PCT Publication No. WO2008/052030 and WO2009126858.

As would be recognized by the skilled person, the constant regions of antibodies also include other constant domains. In particular, these include the CH1 or CL (CK or CA). These immunoglobulin constant domains are also useful in the binding polypeptides described herein. In certain embodiments, junction amino acids are present between an Fc region portion that comprises CH2 and CH3 domains and other immunoglobulin constant region domains that may be used {e.g., a heterodimerization domain such as CH 1 or CL). These junction amino acids are also referred to as a "linker between CH3 and CH 1 or CL" if they are present between the C- terminus of CH3 and the N-terminus of CH 1 or CL. Such a linker may be, for instance, about 2-10 or 12 amino acids in length. In certain embodiments, the Fc region portion comprises human lgG1 CH2 and CH3 domains in which the C-terminal lysine residue of human lgG1 CH3 is deleted. Exemplary linkers between CH3 and CH 1 include those set forth in SEQ ID NO:75-77. Exemplary linkers between CH3 and CK include those set forth in SEQ ID NOS:78-80 (in which the carboxyl terminal arginine in the linkers may alternatively be regarded as the first arginine of CK). In certain embodiments, the presence of such linkers or linker pairs {e.g., SEQ ID NO:75 as a CH3-CH 1 linker in one single chain polypeptide of a heterodimer and SEQ ID NO:78 as a CH3-CK linker in the other single chain polypeptide of the heterodimer; SEQ ID NO:76 as a CH3- CH 1 linker and SEQ ID NO:79 as a CH3-CK linker; and SEQ ID NO:77 as a CH3-CH 1 linker and SEQ ID NO:80 as a CH3-CK linker) improves the production of heterodimer as compared to the presence of a reference linker, such as the reference KSR sequence as set forth in SEQ ID NO:81 in both single chain polypeptides of a heterodimer.

The polypeptides comprising binding domains as described herein may also comprise any of a variety of hinge regions. A hinge region contained in any of the binding polypeptides described herein according to the present disclosure may be located (a) immediately amino terminal to an Fc region portion {e.g., depending on the isotype, amino terminal to a CH2 domain wherein the Fc region portion is a CH2CH3, or amino terminal to a CH3 domain wherein the Fc region portion is a CH3CH4), (b) interposed between and connecting a binding domain {e.g., scFv) and a heterodimerization domain, (c) interposed between and connecting a heterodimerization domain and an Fc region portion {e.g., wherein the Fc region portion is a CH2CH3 or a CH3CH4, depending on the isotype or isotypes), (d) interposed between and connecting an Fc region portion and a binding domain, whether the binding domain is situated at the amino terminal end of the Fc region portion or the carboxyl terminal end of the Fc region portion, or both, (e) at the amino terminus of the single chain polypeptide, or (f) at the carboxyl terminus of the single chain polypeptide.

By way of background, an immunoglobulin hinge acts as a flexible spacer to allow the Fab portion to move freely in space. In contrast to the constant regions, hinges are structurally diverse, varying in both sequence and length between immunoglobulin classes and even among subclasses. For example, a human lgG1 hinge region is freely flexible, which allows the Fab fragments to rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. By comparison, a human lgG2 hinge is relatively short and contains a rigid poly-proline double helix stabilized by four inter-heavy chain disulfide bridges, which restricts the flexibility. A human lgG3 hinge differs from the other subclasses by its unique extended hinge region (about four times as long as the lgG1 hinge), containing 62 amino acids (including 21 prolines and 1 1 cysteines), forming an inflexible poly-proline double helix and providing greater flexibility because the Fab fragments are relatively far away from the Fc fragment. A human lgG4 hinge is shorter than lgG1 but has the same length as lgG2, and its flexibility is intermediate between that of lgG1 and lgG2.

According to crystallographic studies, an IgG hinge region can be functionally and structurally subdivided into three regions: the upper, the core or middle, and the lower hinge regions (Shin et al., Immunological Reviews 130:87 (1992)). Exemplary upper hinge regions include EPKSCDKTHT (SEQ ID NO:37) as found in lgG1 , ERKCCVE (SEQ ID NO:38) as found in lgG2, ELKTPLGDTT HT (SEQ ID NO:39) or EPKSCDTPPP (SEQ ID NO:40) as found in lgG3, and ESKYGPP (SEQ ID NO:41 ) as found in lgG4. Exemplary middle or core hinge regions include CPPCP (SEQ ID NO:42) as found in lgG1 and lgG2, CPRCP (SEQ ID NO:43) as found in lgG3, and CPSCP (SEQ ID NO:44) as found in lgG4. While lgG1 , lgG2, and lgG4 antibodies each appear to have a single upper and middle hinge, lgG3 has four in tandem - one being ELKTPLGDTTHTCPRCP (SEQ ID NO:45) and three being EPKSCDTPPP CPRCP (SEQ ID NO:46).

IgA and IgD antibodies appear to lack an IgG-like core region, and IgD appears to have two upper hinge regions in tandem (see SEQ ID NOS:47 and 48). Exemplary wild type upper hinge regions found in lgA1 and lgA2 antibodies are set forth in SEQ ID NOS:49 and 50.

IgE and IgM antibodies, in contrast, lack a typical hinge region and instead have a CH2 domain with hinge-like properties. Exemplary wild-type CH2 upper hinge-like sequences of IgE and IgM are set forth in SEQ ID NO:51 (VCSRDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTINITWLEDG QVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFE DSTKKCA) and SEQ ID NO:52 (VIAELPPKVSVFVPPRDGFFGNPRKSKLIC QATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTI KESDWLGQSMFTCRVDHRGLTFQQNASSMCVP), respectively.

As used herein, a "hinge region" or a "hinge" refers to (a) an

immunoglobulin hinge region (made up of, for example, upper and core regions) or a functional variant thereof, including wild type and altered immunoglobulin hinges, (b) a lectin interdomain region or a functional variant thereof, (c) a cluster of differentiation (CD) molecule stalk region or a functional variant thereof, or (d) a portion of a cell surface receptor (interdomain region) that connects immunoglobulin V-like or immunoglobulin C-like domains.

As used herein, a "wild type immunoglobulin hinge region" refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody. In certain embodiments, a wild type immunoglobulin hinge region sequence is human, and in certain particular embodiments, comprises a human IgG hinge region. Exemplary human wild type immunoglobulin hinge regions are set forth in SEQ ID NOS:49 (lgA1 hinge), 50 and 329 (lgA2 hinge), 53 (IgD hinge), 54 (lgG1 hinge), 55 (lgG2 hinge), 56 (lgG3 hinge) and 57 (lgG4 hinge).

An "altered wild type immunoglobulin hinge region" or "altered

immunoglobulin hinge region" refers to (a) a wild type immunoglobulin hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (b) a portion of a wild type immunoglobulin hinge region that has a length of about 5 amino acids {e.g., about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) up to about 120 amino acids (for instance, having a length of about 10 to about 40 amino acids or about 15 to about 30 amino acids or about 15 to about 20 amino acids or about 20 to about 25 amino acids), has up to about 30% amino acid changes {e.g., up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1 % amino acid substitutions or deletions or a combination thereof), and has an IgG core hinge region as set forth in SEQ ID NOS:42-44. In certain embodiments, one or more cysteine residues in a wild type or altered immunoglobulin hinge region may be substituted by one or more other amino acid residues {e.g., serine, alanine). In further embodiments, an altered immunoglobulin hinge region may alternatively or additionally have a proline residue substituted by another amino acid residue {e.g., serine, alanine). Exemplary altered wild type immunoglobulin hinge regions include those as set forth in SEQ ID NOS:58-70.

In certain embodiments, a hinge is a wild type human immunoglobulin hinge region {e.g., human immunoglobulin hinge regions as set forth in SEQ ID NOS:461 -466). In certain other embodiments, one or more amino acid residues may be added at the amino- or carboxyl-terminus of a wild type immunoglobulin hinge region as part of a fusion protein construct design. For example, additional junction amino acid residues at the hinge amino-terminus can be "RT," "RSS," "SS", "TG," or "T", or at the hinge carboxyl-terminus can be "SG", or a hinge deletion can be combined with an addition, such as ΔΡ with "SG" added at the carboxyl terminus. Illustrative variant hinges are provided in SEQ ID NOS:71 -74. In certain embodiments, a hinge is an altered immunoglobulin hinge in which one or more cysteine residues in a wild type immunoglobulin hinge region is substituted with one or more other amino acid residues {e.g., serine or alanine). For example, a hinge may be an altered immunoglobulin hinge based on or derived from a wild type human lgG1 hinge as set forth in SEQ ID

NO:120, which from amino terminus to carboxyl terminus comprises the upper hinge region (EPKSCDKTHT, SEQ ID NO:37) and the core hinge region (CPPCP, SEQ ID NO:42). Exemplary altered immunoglobulin hinges include an immunoglobulin human lgG1 hinge region having one, two or three cysteine residues found in a wild type human lgG1 hinge substituted by one, two or three different amino acid residues {e.g., serine or alanine). An altered immunoglobulin hinge may additionally have a proline substituted with another amino acid {e.g., serine or alanine). For example, the above-described altered human lgG1 hinge may additionally have a proline located carboxyl terminal to the three cysteines of wild type human lgG1 hinge region substituted by another amino acid residue {e.g., serine, alanine). In one embodiment, the prolines of the core hinge region are not substituted. Exemplary altered immunoglobulin hinges are set forth in SEQ ID NOS: 121 -136 and 485-495. In one embodiment, an altered lgG1 hinge is an altered human lgG1 hinge in which the first cysteine is substituted by serine. The sequence of this exemplary altered lgG1 hinge is set forth in SEQ ID NO:472, and is referred to as the " human lgG1 SCC-P hinge" or "SCC-P hinge." In certain embodiments, one or more amino acid residues {e.g., "RT," "RSS," or "T") may be added at the amino-or carboxyl-terminus of a mutated immunoglobulin hinge region as part of a fusion protein construct design.

In certain embodiments, a hinge polypeptide comprises or is a sequence that is at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to a wild type immunoglobulin hinge region, such as a wild type human lgG1 hinge, a wild type human lgG2 hinge, or a wild type human lgG4 hinge.

In further embodiments, a hinge may be a hinge that is not based on or derived from an immunoglobulin hinge (i.e., not a wild type immunoglobulin hinge or an altered immunoglobulin hinge). In one embodiment, these types of non-immunoglobulin based hinges are used on or near the carboxyl end (e.g., located carboxyl terminal to Fc region portions) of the polypeptides described herein. Examples for such hinges include peptides from the interdomain or stalk region of type II C-lectins or CD molecules, such as the stalk regions of CD69, CD72, CD94, NKG2A and NKG2D as set forth in SEQ ID NOS:496-501 . Additional exemplary hinges include those as set forth in SEQ ID NOS:502-537.

Alternative hinges that can be used herein are from portions of cell surface receptors (interdomain regions) that connect immunoglobulin V-like or immunoglobulin C-like domains. Regions between Ig V-like domains where the cell surface receptor contains multiple Ig V-like domains in tandem and between Ig C-like domains where the cell surface receptor contains multiple tandem Ig C-like regions are also contemplated as hinges useful in single chain polypeptides of binding polypeptide heterodimers. In certain embodiments, hinge sequences comprising cell surface receptor interdomain regions may further contain a naturally occurring or added motif, such as an IgG core hinge sequence that confers one or more disulfide bonds to stabilize the binding polypeptide heterodimer formation. Examples of hinges include interdomain regions between the Ig V-like and Ig C-like regions of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166, and CD244.

In certain embodiments, hinge sequences have about 5 to 150 amino acids, about 5 to 10 amino acids, about 10 to 20 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 5 to 60 amino acids, about 5 to 40 amino acids, for instance, about 8 to 20 amino acids or about 12 to 15 amino acids. Hinges may be primarily flexible, but may also provide more rigid characteristics or may contain primarily a-helical structure with minimal β-sheet structure. The lengths or the sequences of the hinges may affect the binding affinities of the binding domains to which the hinges are directly or indirectly (via another region or domain, such as a heterodimerization domain) connected as well as one or more activities of the Fc region portions to which the hinges are directly or indirectly connected.

In certain embodiments, hinge sequences are stable in plasma and serum and are resistant to proteolytic cleavage. The first lysine in the lgG1 upper hinge region may be mutated to minimize proteolytic cleavage. For instance, the lysine may be substituted with methionine, threonine, alanine or glycine, or is deleted (see, e.g., SEQ ID NOS:137-192, which may include junction amino acids at the amino terminus, for instance, RT).

In some embodiments, hinge sequences may contain a naturally occurring or added motif such as an immunoglobulin hinge core structure CPPC (SEQ ID NO:594) that confers the capacity to form a disulfide bond or multiple disulfide bonds to stabilize the carboxyl-terminus of a molecule. In other embodiments, hinge sequences may contain one or more glycosylation sites.

Exemplary hinges, including altered immunoglobulin hinges, are set forth in SEQ ID NOS:507-537, 137-192 and 595-724. Additional illustrative hinges, including variant hinges, are set forth in SEQ ID NOs:725-734.

In certain embodiments, the binding polypeptides comprise more than one hinge. For example, a single chain polypeptide having two binding domains, one of which at the amino terminus and the other at the carboxyl terminus, may have two hinges. One hinge may be directly or indirectly [e.g., via a heterodimerization domain) connected to the binding domain at or near the amino terminus, and the other hinge may be connected {e.g., directly connected) to the other binding domain at or near the carboxyl terminus. In certain embodiments, even if a single chain polypeptide has only one binding domain, it may have more than one hinge, for example, at its amino or carboxyl terminus. In certain embodiments, such as where heterodimerization is desired, such a hinge may interact with a corresponding hinge in a second chain of a heterodimer, such as forming one or more interchain disulfide bonds, to facilitate or enhance heterodimerization of the two chains. A hinge (H-l) of a SCP-I of a binding polypeptide heterodimer "corresponds to" a hinge (H-ll) of a SCP-II of the heterodimer when H-l and H-ll are located on the same end of the Fc region portion of their respective single chain polypeptide. For example, a binding polypeptide heterodimer may comprise the following two single chain polypeptides: A first chain polypeptide from amino to carboxyl terminus comprises a first binding domain, CH1 , hinge, CH2, and CH3, and a second chain polypeptide from amino to carboxyl terminus comprises a CK, first hinge, CH2, CH3, second hinge, and a second binding domain. The hinge in the first chain would be regarded as "corresponding" to the first hinge of the second chain because both are amino terminal to the Fc region portions to which they are connected.

In certain embodiments, particularly where a binding polypeptide comprises a binding domain at or near its carboxyl terminus, a hinge may be present to link the binding domain with another portion of the polypeptide {e.g., an Fc region portion or a heterodimerization domain). In certain embodiments, such a hinge is a non-immunoglobulin hinge (i.e., a hinge not based on or derived from a wild type immunoglobulin hinge) and may be a stalk region of a type II C-lectin or CD molecule, an interdomain region that connect IgV-like or IgC-like domains of a cell surface receptor, or a derivative or functional variant thereof. Exemplary carboxyl terminal hinges, sometimes referred to as "back- end" hinges, include those set forth in SEQ ID NOS: 502, 507-537, 475-714.

In certain embodiments, the polypeptides comprising binding domains as described herein may contain one or more additional domains or regions. Such additional regions may be a leader sequence (also referred to as "signal peptide") at the amino-terminus for secretion of an expressed polypeptide. Exemplary leader peptides of this disclosure include natural leader sequences or others, such as those as set forth in SEQ ID NOS:735 and 736. In one embodiment, the polypeptides of the present invention make use of mature proteins that do not include the leader peptide (signal peptide). Accordingly, while certain sequences provided herein for binding domain proteins (such as for slL6xR) include the leader peptide, the skilled person would readily understand how to determine the mature protein sequence from sequences including a signal peptide. In certain embodiments, it may be useful to include the leader sequence.

Additional regions may also be sequences at the carboxyl-terminus for identifying or purifying single chain polypeptides (e.g., epitope tags for detection or purification, such as a histidine tag, biotin, a FLAG® epitope, or any combination thereof).

Further optional regions may be additional amino acid residues (referred to as "junction amino acids" or "junction amino acid residues") having a length of 1 to about 8 amino acids {e.g., about 2 to 5 amino acids), which may be resulted from use of specific expression systems or construct design for the polypeptides of the present disclosure. Such additional amino acid residues (for instance, about one, two, three, four or five additional amino acids) may be present at the amino or carboxyl terminus or between various regions or domains, such as between a binding domain and a heterodimerization domain, between a heterodimerization domain and a hinge, between a hinge and an Fc region portion, between domains of an Fc region portion {e.g., between CH2 and CH3 domains or between two CH3 domains), between a binding domain and a hinge, or between a variable domain and a linker. Exemplary junction amino acids amino-terminal to a hinge include RDQ (SEQ ID NO:737), RT, SS, SASS (SEQ ID NO:738) and SSS (SEQ ID NO:739). Exemplary junction amino acids carboxyl-terminal to a hinge include amino acids SG. Additional exemplary junction amino acids include SR.

The polypeptides comprising binding domains of the present disclosure may also comprise linkers between any of the various domains as described herein. Exemplary linkers may include any of the linkers as provided in SEQ ID NOS:33, 75-81 , 193-328, 330-362 and 745-748. Illustrative linkers useful in linking the carboxyl terminus of a CH3 domain with an amino terminus of a CH1 or CK domain are provided in SEQ ID NOS:75-81 and 733.

A "peptide linker" or "variable domain linker" refers to an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a similar or better specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions. In certain embodiments, a variable domain linker is comprised of about five to about 35 amino acids and in certain embodiments, comprises about 15 to about 25 amino acids. An illustrative variable domain linker is the (G4S)3 linker as provided in SEQ ID NO:33.

In certain embodiments, there may be one or more {e.g., about 2-8) amino acid residues between a hinge and an Fc region portion due to construct design of fusion polypeptides {e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion polypeptides). As described herein, such amino acid residues may be referred to as "junction amino acids" or "junction amino acid residues." Exemplary junction amino acids are shown in the hinge variant sequences provided in SEQ ID NOS:71 -74 {e.g., in SEQ ID NO:71 , the C- terminal SG residues are considered junction amino acids; in SEQ ID NO:72, the N-terminal SS residues are considered junctional residues; in SEQ ID NO:73, the N-terminal SS and the C-terminal SG residues are considered junction amino acids; in SEQ ID NO:74, the N-terminal RT and the C-terminal SG are junction amino acids).

In certain embodiments, a fusion polypeptide comprising a binding domain as described herein may comprise a single binding domain, a hinge, and an effector domain {e.g., an Fc region constant domain portion), such as a "small modular immunopharmaceutical" (SMIP). These single binding domain binding polypeptides comprise a single polypeptide chain including a target- specific binding domain, based, for example, upon an antibody variable domain, in combination with an Fc region that permits the specific recruitment of a desired class of effector cells (such as, e.g., macrophages and natural killer (NK) cells) and/or recruitment of complement-mediated killing. Depending upon the choice of target and hinge regions, binding polypeptides having this format can signal or block signaling via cell surface receptors. Generally these single binding domain binding proteins are binding domain-immunoglobulin fusion proteins that typically comprise from their amino termini to carboxyl termini: a binding domain derived from an immunoglobulin {e.g., a scFv), a hinge region, and an effector domain {e.g., IgG CH2 and CH3 regions). As used herein, "small modular immunopharmaceutical" or "SMIP products", are as generally described in US Patent Publication Nos. 2003/133939, 2003/01 18592, and 2005/0136049, and International Patent Publications WO02/056910,

WO2005/037989, and WO2005/017148. Two identical SMIPs may form a homodimer with each other.

In some embodiments, a binding polypeptide of the invention comprising a slL6xR binding domain may comprise, from the amino termini to carboxyl termini: an effector and/or constant domain {e.g., IgG CH2 and CH3 regions), a hinge region and a binding domain derived from an immunoglobulin {e.g., a scFv). This molecule is also referred to as a PIMS molecule and examples of PIMS are described in US Patent Publication No. 2009/0148447 and

International Patent Publication WO2009/023386.

In some embodiments, a binding polypeptide of the invention may comprise, from the amino termini to carboxyl termini: a hinge region, an effector and/or constant domain {e.g., IgG CH2 and CH3 regions) and a slL6xR binding domain, e.g., derived from an immunoglobulin {e.g., a scFv).

It should be noted that a primary target of the binding polypeptides of this disclosure is the slL6xR complex, e.g., a human native slL6xR complex.

However, in certain embodiments, the binding polypeptides may comprise one or more additional binding domains that bind slL6xR, or a target other than slL6xR {e.g., heterologous target including but not limited to TNFR and TNFa). These heterologous target molecules may comprise, for example, a particular cytokine or a molecule that targets the binding domain polypeptide to a particular cell type, a toxin, an additional cell receptor, an antibody, a cytokine, etc.

Thus, another aspect of the present disclosure provides binding polypeptides that comprise a binding domain that binds to slL6xR and further comprises a second binding domain that binds to another target protein. Such multi-specific proteins may be as described for example in published US application 201 1/0152173 or 201 1/0177070 or in published PCT application WO 201 1/079308. Other target proteins contemplated as targets for use binding polypeptides or binding domains described herein include, but are not limited to, a TNFa {e.g., binding domains that are TNFa antagonists, such as a TNFa binding domain or a TNF receptor (TNFR) ectodomain (such as those described in WO 2010/003108)), a TGF (binding domains such as those described in WO 2010/0031 18), a tumor antigen, a B-cell target, a TNF receptor superfamily member, a Hedgehog family member, a receptor tyrosine kinase, a proteoglycan-related molecule, a TGF-beta superfamily member, a Wnt-related molecule, a receptor ligand, a T-cell target, a dendritic cell target, an NK cell target, a monocyte/macrophage cell target and an angiogenesis target.

TNFRs are type I transmembrane proteins having an extracellular domain that contains three well ordered cysteine rich domains (CRD1 , CRD2, CRD3) characteristic of the TNFR superfamily, and a fourth less well conserved, membrane proximal CRD (Banner et al. (1993) Cell 73:431 ). A TNF-a antagonist of this disclosure inhibits the inflammatory or

hyperproliferative activity of TNF-a. Antagonist domains may block TNFR multimerization or TNF-a binding, or the domains may bind to components of the receptor system and block activity either by preventing ligand activity or by preventing the assembly of the receptor complex. Various TNF-a antagonists are known in the art and can be used in accordance with this disclosure, including, but not limited to, anti-TNF antibodies, such as infliximab and

Adalimumab (HUMIRA^®) or binding domains derived therefrom (such as scFvs) and soluble TNF receptors (sTNFR). Such antibody antagonists bind and inhibit TNF-a, but do not significantly inhibit TNF-β. Anti-TNF antibodies, including monoclonal antibodies, can be prepared using known techniques and are known in the art (see, e.g., US Patent No. 6,509,015). A TNF-a antagonist of this disclosure can also comprise one or more TNF-a binding domains present in a TNFR ectodomain.

In some embodiments, a TNF-a antagonist includes a TNFR

extracellular domain or sub-domain, one or more TNFR CRD domains (such as CRD2 and CRD3), or TNF-a-specific antibody-derived binding domains

(analogous to the slL6xR-specific antibody-derived binding domain described herein). In some embodiments, a TNF-a antagonist domain is a binding domain from or derived from an antibody such as a scFv. In some

embodiments, a TNF-a binding domain comprises amino acids 23-141 of SEQ ID NO:754, amino acids 159-265 of SEQ ID NO:754, amino acids 23-141 and 159-265 of SEQ ID NO:754 or amino acids 23-265 of SEQ ID NO:754. In some embodiments, a TNF-a antagonist may be an extracellular domain

("ectodomain") of a TNFR, such as an ectodomain of TNFR1 or TNFR2. As used herein, a TNFR ectodomain refers to a sTNFR, one or more CRDs, or any combination thereof of the TNFR domains. In certain embodiments, a TNF-a antagonist comprises an amino-terminal portion of TNFR2 (also known as p75, TNFRSF1 B), such as the first 257 amino acids of TNFR2 as set forth in

GenBank Accession No. NP_001057.1 (SEQ ID NO:1 18). In other

embodiments, a TNF-a antagonist comprises amino acids 23-257 of SEQ ID NO:1 18 (i.e., without the native leader sequence). In some embodiments, a TNF-a antagonist comprises a fragment of TNFR2 (e.g. , an ectodomain), such as amino acids 23-163 of SEQ ID NO:1 18 or amino acids 23-185 of SEQ ID NO:1 18 or amino acids 23-235 of SEQ ID NO:1 18. In some embodiments, a TNF-a antagonist comprises a derivative of a TNFR2 fragment, such as amino acids 23-163 of SEQ ID NO:1 18 with a deletion of amino acid glutamine at position 109 or amino acids 23-185 of SEQ ID NO:1 18 with a deletion of amino acid glutamine at position 109 and a deletion of amino acid proline at position 131 or amino acids 23-235 of SEQ ID NO:1 18 with a deletion of amino acid glutamine at position 109, a deletion of amino acid proline at position 131 , and a substitution of amino acid aspartate at position 257 (to, for example, a threonine, alanine, serine, or glutamate). In further embodiments, a TNF-a antagonist comprises an amino-terminal portion of TNFR1 (also known as p55, TNFRSF1A), such as the first 21 1 amino acids of TNFR1 as set forth in

GenBank Accession No. NP_001056.1 (SEQ ID NO:1 19). In other

embodiments, a TNF-a antagonist comprises amino acids 31 -21 1 of SEQ ID NO:1 19 (i.e., without the native leader sequence).

In certain embodiments a slL6xR binding domain of the invention may be present within a binding polypeptide such as those described in PCT

application No. WO2007/146968, US2009/059446 and U.S. Patent Application Publication No. 2006/0051844. Polypeptides may comprise a slL6xR binding domain and a binding domain that binds a molecule other than slL6xR

("heterologous binding domain" also referred to as a binding domain which binds to a heterologous target protein). In certain embodiments, the

heterologous binding domain specifically binds to a target molecule including, but not limited to TNFa, a tumor antigen, a B-cell target, a TNF receptor superfamily member, a Hedgehog family member, a receptor tyrosine kinase, a proteoglycan-related molecule, a TGF-beta superfamily member, a Wnt-related molecule, a receptor ligand, a T-cell target, a Dendritic cell target, an NK cell target, a monocyte/macrophage cell target or an angiogenesis target. In this regard, the heterologous binding domain may comprise an immunoglobulin- based binding domain (e.g., an scFv) or may comprises a receptor ECD (e.g., TNFR ectodomain, type 1 insulin-like growth factor receptor 1 (IGF1 R ectodomain)).

In certain embodiments, the present disclosure provides binding polypeptides that bind both an IL6xR complex and a second target such as, but not limited to, a receptor activator of nuclear factor kappa B ligand (RANKL, also known as TNFSF1 1 , ODF, CD254), IL7, IL17A, IL17F, IL17A/F,

Glucocorticoid-induced tumor necrosis factor receptor (GITR), VEGF, TNF, HGF, type 1 insulin-like growth factors IGF1 or IGF2, Tumor necrosis factor-like weak inducer of apoptosis (TWEAK; also known as tumor necrosis factor (ligand) superfamily, member 12, TNFSF12), colony stimulating factor 2 (CSF2, also known as granulocyte-macrophage colony stimulating factor or GM-CSF); insulin-like growth factor-1 (IGF1 ); insulin-like growth factor-2 (IGF2); IL10; or a TNFSF13 family protein {e.g., TNFSF13, also known as a proliferation-inducing ligand, APRIL, CD256; or TNFSF13B, also known as B-lymphocyte stimulator, BLyS, CD257, BAFF).

In one embodiment, the heterologous binding domain may bind a

Transforming Growth Factor (TGF)-beta superfamily member selected from the group consisting of Activin Rl A/ ALK-2, GFR alpha-1 , Activin RIB/ALK-4, GFR alpha-2, Activin RIIA, GFR alpha-3, Activin RUB, GFR alpha-4, ALK-I, MIS Rll, ALK-7, Ret, BMPR-I A/ALK-3, TGF-beta RI/ALK-5, BMPR-IB/ALK-6, TGF-beta Rll, BMPR-II, TGF-beta Rllb, Endoglin/CD105 and TGF-beta RIM.

In one embodiment, the heterologous binding domain may bind a target tumor antigen selected from the group consisting of squamous cell carcinoma antigen 1 (SCCA-I), (protein t4-a), squamous cell carcinoma antigen 2 (SCCA- 2), ovarian carcinoma antigen CA125 (1 a1 -3b) (KIAA0049), mucin 1 (tumor- associated mucin), (carcinoma-associated mucin), (polymorphic epithelial mucin), (pem),(pemt),(episialin), (tumor-associated epithelial membrane antigen), (EMA),(h23 AG), (peanut-reactive urinary mucin), (PUM), (breast carcinoma-associated antigen DF3), CTCL tumor antigen sel-1 , CTCL tumor antigen sel4-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-l, CTCL tumor antigen se37-2, CTCL tumor antigen se57-l, CTCL tumor antigen se89-l, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-CI (cancer/testis antigen CT7), MAGE-BI ANTIGEN (MAGE-XP

ANTIGEN) (DAMIO), MAGE-B2 ANTIGEN (DAM6), MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE -4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen,

Adenocarcinoma antigen ARTI, Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen), Neuro- oncological ventral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, tumor-associated antigen CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1 ,

Serologically defined breast cancer antigen NY-BR-15, Serologically defined breast cancer antigen NY-BR-16, Chromogranin A; parathyroid secretory protein 1 , DUPAN-2, CA 19-9, CA 72-4, CA 195 and L6.

In one embodiment, the heterologous binding domain may bind a B cell target selected from the group consisting of CD10, CD19, CD20, CD21 , CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81 , CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDwl30, CD138 and CDwl50. In other embodiments of the above-described method, the TNF receptor superfamily member is selected from the group consisting of 4-1 BB/TNFRSF9, NGF R/TNFRSF16, BAFF R/TNFRSF13C, Osteoprotegerin/TNFRSFI IB,

BCMA/TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7, RANK/TNFRSF1 1 A, CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF5, TACI/TNFRSF13B, DcR3/TNFRSF6B, TNF RI/TNFRSF1 A, DcTRAIL R1/TNFRSF23, TNF

RII/TNFRSF1 B, DcTRAIL R2/TNFRSF22, TRAIL R1/TNFRSF10A,

DR3/TNFRSF25, TRAIL R2/TNFRSF10B, DR6/TNFRSF21 , TRAIL

R3/TNFRSF10C, EDAR, TRAIL R4/TNFRSF10D, Fas/TNFRSF6,

TROY/TNFRSF19, GITR/TNFRSF18, TWEAK R/TNFRSF12,

HVEM/TNFRSF14, XEDAR, Lymphotoxin beta R/TNFRSF3, 4-IBB

Ligand/TNFSF9, Lymphotoxin, APRIL/TNFSF13, Lymphotoxin beta/TNFSF3, BAFF/TNFSF13C, OX40 Ligand/TNFSF4, CD27 Ligand/TNFSF7,

TL1A/TNFSF15, CD30 Ligand/TNFSF8, TNF-alpha/TNFSFIA, CD40

Ligand/TNFSF5, TNF-beta/TNFSFIB, EDA-A2, TRAIL/TNFSF 10, Fas

Ligand/TNFSF[beta], TRANCE/TNFSF 1 1 , GITR Ligand/TNFSF18,

TWEAK/TNFSF12 and LIGHT/TNFSF14. In certain embodiments, the receptor tyrosine kinase is selected from the group consisting of Axl, FGF R4, Clq R1/CD93, FGF R5, DDRI, Flt-3, DDR2, HGF R, Dtk, IGF-I R, EGF R, IGF-II R, Eph, INSRR, EphAI, Insulin R/CD220, EphA2, M-CSF R, EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R alpha, EphA7, PDGF R beta, EphA8, Ret, EphBI, RORI, EphB2, ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1 , EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF Rl, VEGF RI/Flt-1 , FGF R2, VEGF R2/Flk-1 , FGF R3 and VEGF R3/Flt-4.

In yet other embodiments, the binding polypeptides described herein comprise a heterologous binding domain which binds to a Wnt-related molecule selected from the group consisting of Frizzled-1 , Frizzled-8, Frizzled-2, Frizzled- 9, Frizzled-3 , sFRP-1 , Frizzled-4, sFRP-2, Frizzled-5 , sFRP-3 , Frizzled-6, sFRP-4, Frizzled-7, MFRP, LRP 5, LRP 6, Wnt-1 , Wnt-8a, Wnt-3a, Wnt-IOb, Wnt-4, Wnt-1 1 , Wnt-5a, Wnt-9a and Wnt-7a.

In other embodiments the binding polypeptides described herein comprise a heterologous binding domain which binds to a receptor ligand selected from the group consisting of 4-IBB Ligand/TNFSF9, Lymphotoxin, APRIL/TNFSF13, Lymphotoxin beta/TNFSF3, BAFF/TNFSF13C, OX40

Ligand/TNFSF4, CD27 Ligand/TNFSF7, TL1 A/TNFSF15, CD30

Ligand/TNFSF8, TNF-alpha/TNFSFIA, CD40 Ligand/TNFSF5, TNF- beta/TNFSFIB, EDA-A2, TRAIL/TNFSF10, Fas Ligand/TNFSF[beta],

TRANCE/TNFSF1 1 , GITR Ligand/TNFSF18, TWEAK/TNFSF12,

LIGHT/TNFSF14, Amphiregulin, NRGI isoform GGF2, Betacellulin, NRGI Isoform SMDF, EGF, NRG 1 -alpha/HRGI -alpha, Epigen, NRGI-beta 1/HRGI- beta 1 , Epiregulin, TGF-alpha, HB-EGF, TMEFF 1/Tomoregulin-l, Neuregulin-3, TMEFF2, IGF-I, IGF-II, Insulin, Activin A, Activin B, Activin AB, Activin C, BMP- 2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4, BMP-15, BMP-5, Decapentaplegic, BMP-6, GDF-I, GDF-8, GDF-3, GDF-9, GDF-5, GDF-I I, GDF-6, GDF-15, GDF-7, Artemin, Neurturin, GDNF, Persephin, TGF-beta, TGF-beta 2, TGF-beta 1 , TGF-beta 3, LAP (TGF-beta 1 ), TGF-beta 5, Latent TGF-beta 1 , Latent TGF-beta bpl, TGF-beta 1 .2, Lefty, Nodal, M IS/AM H, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21 , FGF-9, FGF-23, FGF-IO, KGF/FGF- 7, FGF-I I, Neuropilin-1 , PIGF, Neuropilin-2, P1 GF-2, PDGF, PDGF-A, VEGF, PDGF-B, VEGF-B, PDGF-C, VEGF-C, PDGF-D, VEGF-D and PDGF-AB.

In still other embodiments, the binding polypeptides described herein comprise a heterologous binding domain which binds to a T-cell target selected from the group consisting of 2B4/SLAMF4, IL-2 R alpha, 4-1 BB/TNFRSF9, IL-2 R beta, ALCAM, B7-1/CD80, IL-4 R, B7-H3, BLAME/SLAMF8, BTLA, IL-6 R, CCR3, IL-7 R alpha, CCR4, CXCRI/IL-8 RA, CCR5, CCR6, IL-10 R alpha, CCR7, IL-10 R beta, CCR8, IL-12 R beta 1 , CCR9, IL-12 R beta 2, CD2, IL-13 R alpha 1 , IL-13, CD3, CD4, ILT2/CD85J, ILT3/CD85k, ILT4/CD85d,

ILT5/CD85a, Integrin alpha 4/CD49d, CD5, Integrin alpha E/CD103, CD6, Integrin alpha M/CD1 lb, CD8, Integrin alpha X/CDI lc, Integrin beta 2/CD18, KIR/CD158, CD27/TNFRSF7, KIR2DL1 , CD28, KIR2DL3, CD30/TNFRSF8, KIR2DL4/CD158d, CD31/PECAM-1 , KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIRI, CD45, LAIR2, CD83, Leukotriene B4 Rl, CD84/SLAMF5, NCAM- Ll, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F- 10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common gamma Chain/IL-2 R gamma, Osteopontin, CRACC/SLAMF7, PD-I, CRTAM, PSGL-I, CTLA-4, RANK/TNFRSF1 1 A, CX3CR1 , CX3CL1 , L-Selectin, CXCR3, SIRP beta 1 , CXCR4, SLAM, CXCR6, TCCR/WSX-1 , DNAM-I, Thymopoietin,

EMMPRIN/CD147, TIM-I, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas

Ligand/TNFSF[beta], TIM-4, Fc gamma RIII/CD16, TIM-6, GITR/TNFRSF18, TNF RI/TNFRSF1A, Granulysin, TNF RII/TNFRSF1 B, HVEM/TNFRSF14, TRAIL R1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM- 2/CD102, TRAIL R3/TNFRSF10C, IFN-gamma Rl, TRAIL R4/TNFRSF10D, IFN-gamma R2, TSLP, IL-I Rl and TSLP R.

In other embodiments, the binding polypeptides described herein comprise a heterologous binding domain which binds to a NK cell receptor selected from the group consisting of 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1 , CD94, LMIR1/CD300A, CD69, LMIR2/CD300C, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-I, LMIR5/CD300LB, Fc epsilon Rll, LMIR6/CD300LE, Fc gamma RI/CD64, MICA, Fc gamma RIIB/CD32b, MICB, Fc gamma

RIIC/CD32C, MULT-1 , Fc gamma RIIA/CD32a, Nectin-2/CDI 12, Fc gamma RIII/CD16, NKG2A, FcRHI/IRTA5, NKG2C, FcRH2/IRTA4, NKG2D,

FcRH4/IRTAI, NKp30, FcRH5/IRTA2, NKp44, Fc Receptor-like 3/CD16-2,

NKp46/NCRI, NKp80/KLRFI, NTB-A/SLAMF6, Rae-1 , Rae-1 alpha, Rae-1 beta, Rae-1 delta, H60, Rae-1 epsilon, ILT2/CD85J, Rae-1 gamma, ILT3/CD85k, TREM-I, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158,

TREMLI/TLT-1 , KIR2DL1 , ULBP-I, KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3.

In other embodiments, the binding polypeptides described herein comprise a heterologous binding domain which binds to a

monocyte/macrophage cell target selected from the group consisting of B7- 1/CD80, ILT4/CD85d, B7-H1 , ILT5/CD85a, Common beta Chain, Integrin alpha 4/CD49d, BLAME/SLAMF8, Integrin alpha X/CD 1 1 c, CCL6/C10, Integrin beta 2/CD18, CD155/PVR, Integrin beta 3/CD61 , CD31/PECAM-1 , Latexin,

CD36/SR-B3, Leukotriene B4 Rl, CD407TNFRSF5, LIMPII/SR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300C, CD68, LMIR3/CD300LF,

CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD 163, LRP-I, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-I, ECF-L, MD-2,

EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB, Fc gamma RI/CD64, Osteopontin, Fc gamma RIIB/CD32b, PD-L2, Fc gamma RIIC/CD32c, Siglec-3/CD33, Fc gamma RIIA/CD32a, SIGNR1/CD209, Fc gamma RIII/CD16, SLAM, GM-CSF R alpha, TCCR/WSX-1 , ICAM-2/CD102, TLR3, IFN-gamma Rl, TLR4, IFN-gamma R2, TREM-I, IL-I Rll, TREM-2, ILT2/CD85J, TREM-3, ILT3/CD85k, TREMLI/TLT-1 , 2B4/SLAMF4, IL-IO R alpha, ALCAM, IL-10 R beta, Aminopeptidase N/ANPEP, ILT2/CD85J , Common beta Chain,

ILT3/CD85k, Clq R1/CD93, ILT4/CD85d, CCRI, ILT5/CD85a, CCR2, Integrin alpha 4/CD49d, CCR5, Integrin alpha M/CD1 lb, CCR8, Integrin alpha X/CD1 lc, CD155/PVR, Integrin beta 2/CD18, CD14, Integrin beta 3/CD61 , CD36/SR- B3, LAIRI, CD43, LAIR2, CD45, Leukotriene B4 Rl, CD68, LIMPII/SR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300C, CD163,

LMIR3/CD300LF, Coagulation Factor Ill/Tissue Factor, LMIR5/CD300LB, CX3CR1 , CX3CL1 , LMIR6/CD300LE, CXCR4, LRP-I, CXCR6, M-CSF R, DEP- 1/CD148, MD-I, DNAM-I, MD-2, EMMPRIN/CD147, MMR, Endoglin/CD105, NCAM-LI, Fc gamma RI/CD64, PSGL-I, Fc gamma RIII/CD16, RP 105, G-CSF R, L-Selectin, GM-CSF R alpha, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1 , ICAM-2/CD102, TREM-I, IL-6 R, TREM-2, CXCRI/IL-8 RA, TREM-3 and TREML 1/TLT-l.

In yet other embodiments the binding polypeptides described herein comprise a heterologous binding domain which binds to a Dendritic cell target selected from the group consisting of CD36/SR-B3, LOX-1/SR-EI, CD68, MARCO, CD163, SR-AI/MSR, CD5L, SREC-I, CL-P1/COLEC12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLRI, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB Ligand/TNFSF9, IL-12/IL-23 p40, 4-Amino-l,8-naphthalimide, ILT2/CD85J, CCL21/6Ckine, ILT3/CD85k, 8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, Integrin alpha 4/CD49d, Aag, Integrin beta 2/CD18, AMICA, Langerin, B7-2/CD86, Leukotriene B4 Rl, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300C, Clq R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB, CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4a, CD43, MCAM, CD45, MD-I, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAM-LI, CD2F-10/SLAMF9, Osteoactivin/GPNMB, Chem 23, PD-L2, CLEC-I, RP 105, CLEC-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5, DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7,

DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1/CLEC7A, Siglec-F, Dectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1 , Fc gamma RI/CD64, TLR3, Fc gamma RIIB/CD32b, TREM-I, Fc gamma RIIC/CD32c, TREM-2, Fc gamma

RIIA/CD32a, TREM-3, Fc gamma RIII/CD16, TREMLI/TLT-1 , ICAM-2/CD102 and Vanilloid Rl.

In still other embodiments, the binding polypeptides described herein comprise a heterologous binding domain which binds to an angiogenesis target selected from the group consisting of Angiopoietin-1 , Angiopoietin-like 2, Angiopoietin-2, Angiopoietin-like 3, Angiopoietin-3, Angiopoietin-like 7/CDT6, Angiopoietin-4, Tie-1 , Angiopoietin-like 1 , Tie-2, Angiogenin, iNOS, Coagulation Factor Ill/Tissue Factor, nNOS, CTGF/CCN2, NOV/CCN3, DANCE, OSM, EDG-I, Plfr, EG-VEGF/PK1 , Proliferin, Endostatin, ROBO4, Erythropoietin, Thrombospondin-1 , Kininostatin, Thrombospondin-2, MFG-E8,

Thrombospondin-4, Nitric Oxide, VG5Q, eNOS, EphAI, EphA5, EphA2, EphA6, EphA3, EphA7, EphA4, EphA8, EphBI, EphB4, EphB2, EphB6, EphB3, Ephrin- Al, Ephrin-A4, Ephrin-A2, Ephrin-A5, Ephrin-A3, Ephrin-BI, Ephrin-B3, Ephrin- B2, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21 , FGF-9, FGF-23, FGF-IO, KGF/FGF-7, FGF-I I, FGF Rl, FGF R4, FGF R2, FGF R5, FGF R3, Neuropilin- 1 , Neuropilin-2, Semaphorin 3 A, Semaphorin 6B, Semaphorin 3C, Semaphorin 6C, Semaphorin 3E, Semaphorin 6D, Semaphorin 6A, Semaphorin 7A, MMP, MMP-1 1 , MMP-I, MMP-12, MMP-2, MMP-13, MMP-3, MMP-14, MMP-7, MMP- 15, MMP-8, MMP-16/MT3-MMP, MMP-9, MMP-24/MT5-MMP, MMP-IO, MMP- 25/MT6-MMP, TIMP-I, TIMP-3, TIMP-2, TIMP-4, ACE, IL-13 R alpha 1 , IL-13, Clq R1/CD93, Integrin alpha 4/CD49d, VE-Cadherin, Integrin beta 2/CD18, CD31/PECAM-1 , KLF4, CD36/SR-B3, LYVE-I, CD151 , MCAM, CL- P1/COLEC12, Nectin-2/CDI 12, Coagulation Factor Ill/Tissue Factor, E-

Selectin, D6, P-Selectin, DC-SIGNR/CD299, SLAM, EMMPRIN/CD147, Tie-2, Endoglin/CD105, TNF RI/TNFRSF1A, EPCR, TNF RII/TNFRSF1 B,

Erythropoietin R, TRAIL R1/TNFRSF10A, ESAM, TRAIL R2/TNFRSF10B, FABP5, VCAM-I, ICAM-1/CD54, VEGF R2/Flk-1 , ICAM-2/CD102, VEGF R3/Flt- 4, IL-I Rl and VG5Q.

In other embodiments, the binding polypeptides described herein comprise a heterologous binding domain which binds to a target selected from the group consisting of Prostate-specific Membrane Antigen (Folate Hydrolase 1 ), Epidermal Growth Factor Receptor (EGFR), Receptor for Advanced

Glycation End products (RAGE, also known as Advanced Glycosylation End product Receptor or AGER), IL-17 A, IL-17 F, PI 9 (IL23A and IL 12B), Dickkopf-1 (Dkkl), NOTCHI, NG2 (Chondroitin Sulfate ProteoGlycan 4 or CSPG4), IgE (IgHE or lgH2), IL-22R (IL22RA1 ), IL-21 , Amyloid [beta] oligomers (Ab oligomers), Amyloid [beta] Precursor Protein (APP), NOGO Receptor (RTN4R), Low Density Lipoprotein Receptor-Related Protein 5 (LRP5), IL-4, Myostatin (GDF8), Very Late Antigen 4, an alpha 4, beta 1 integrin (VLA4 or ITGA4), an alpha 4, beta 7 integrin found on leukocytes.

In certain embodiments, the binding polypeptides described herein comprise a heterologous binding domain which binds to a myeloid target, including but not limited to, CD5, CDIO, CDI lb, CDI lc, CD13, CD14, CD15, CD18, CD19, CD20, CD21 , CD22, CD23, CD25, CD27, CD29, CD30, CD31 , CD33, CD34, CD35, CD38, CD43, CD45, CD64, CD66, CD68, CD70, CD80, CD86, CD87, CD88, CD89, CD98, CD100, CD103, CDI I I, CDI 12, CDI 14, CDI 15, CDI 16, CDI 17, CDI 18, CDI 19, CD120a, CD120b, CDwl23, CDwl31 , CD141 , CD162, CD163, CD177, CD312, IRTAI, IRTA2, IRTA3, IRTA4, IRTA5, B-B2, B-B8 and B-cell antigen receptor.

It is contemplated that a slL6xR binding domain may be at the amino- terminus and the heterologous binding domain at the carboxyl-terminus of a multi-specific binding polypeptide fusion protein. It is also contemplated that a heterologous binding domain may be at the amino-terminus and the slL6xR binding domain may be at the carboxyl-terminus. As set forth herein, the binding domains of this disclosure may be fused to each end of an intervening domain {e.g., an immunoglobulin constant region such as an Fc region constant domain). Furthermore, the two or more binding domains may be each joined to an intervening domain via a linker, as described herein.

As used herein, an "intervening domain" refers to an amino acid sequence that simply functions as a scaffold for one or more binding domains so that an isolated polypeptide or fusion protein will exist primarily {e.g., about 50% or more of a population of fusion proteins) or substantially {e.g., about 90% or more of a population of fusion proteins) as a single chain polypeptide in a composition. For example, certain intervening domains can have a structural function {e.g., spacing, flexibility, rigidity) or biological function {e.g., an increased half-life in plasma, such as in human blood). Exemplary intervening domains that can increase half-life of the polypeptides of this disclosure in plasma include albumin, transferrin, a scaffold domain that binds a serum protein, or the like, or fragments thereof.

In certain embodiments, the intervening domain contained in a multi- specific fusion protein of this disclosure is a dimerization domain as described elsewhere herein. In certain embodiments, two identical multi-specific fusion proteins may form a homodimer with each other.

Exemplary structures of multi-specific polypeptides comprising a slL6xR binding domain include N-BD1 -X-BD2-C and N-BD2-X-BD1 -C, wherein N and C represent the amino-terminus and carboxyl-terminus, respectively; BD1 is a slL6xR binding domain, such as an immunoglobulin-like or immunoglobulin variable region binding domain; X is an intervening domain, and BD2 is a binding domain that is a heterologous binding domain, i.e., a binding domain that binds a protein other than slL6xR, such as, but not limited to, TNFa, TGF , or other heterologous binding domain as described herein. In certain

embodiments, both BD1 and BD2 are immunoglobulin-like or immunoglobulin variable region binding domains, and the polypeptides may also be referred to as "Scorpion" proteins. In some constructs, X can comprise an immunoglobulin constant region disposed between the first and second binding domains. In some embodiments, a binding polypeptide has an intervening domain (X) comprising, from amino-terminus to carboxyl-terminus, a structure as follows: - L1 -X-L2-, wherein L1 and L2 are each independently a linker comprising from about two to about 150 amino acids; and X is an immunoglobulin constant domain. In certain embodiments, the L1 and L2 linkers may be hinges as described herein, and in some embodiments the L2 linker is derived from an interdomain region of an immunoglobulin superfamily member or an

extracellular stalk region of a type 2 membrane protein in the C-type lectin family. In further embodiments, the binding polypeptide will have an

intervening domain that is albumin, transferrin, or another serum protein binding protein, wherein the fusion protein remains primarily or substantially as a single chain polypeptide in a composition.

In still further embodiments, a binding polypeptide of this disclosure has the following structure: N-BD1 -X-L2-BD2-C, wherein BD1 is a slL6xR binding domain, such as a binding domain that is at least about 90% identical to a slL6xR binding domain, such as an scFv comprising the VH having amino acid sequences set forth in SEQ ID NOS:13, 19, 24 and 29; and VL having amino acid sequences set forth in SEQ ID NOs:14, 20, 25 and 30; -X- is

-L1 -CH2CH3-, wherein L1 is a first lgG1 hinge, optionally mutated by

substituting the first or second cysteine and wherein -CH2CH3- is the CH2CH3 region of an lgG1 Fc domain; L2 is a linker selected from SEQ ID NOS:193-360 and 745-748; and BD2 is a heterologous binding domain that binds to a molecule other than slL6xR.

In certain embodiments, the present disclosure provides a multi-valent binding molecule that comprises multiple slL6xR binding domains. In one embodiment, multiple slL6xR binding domains may be linked in tandem and function as BD1 or BD2 as described in the structures herein above. In another embodiment, both binding domains of the multi-specific binding molecule may be slL6xR binding domains {e.g., both BD1 and BD2 are slL6xR binding domains). In embodiments where both BD1 & BD2 are slL6xR binding domains, the binding domains can be the same slL6xR binding domains or different slL6xR binding domains.

As noted above, the binding polypeptides disclosed herein may be in the form of an antibody or a fusion protein of any of a variety of different formats {e.g., the fusion protein may comprise the format of a binding domain- hinge/linker-Fc such as in polypeptide as described in U.S. Patent Publication Nos. 2003/0133939, 2003/01 18592, and 2005/0136049 or PCT Application Publication No. WO 2009/023386. In another embodiment, the binding polypeptide may be in a format N-BD1 -X-L2-BD2-C wherein BD1 comprises an scFv specific for an IL-6/slL-6R complex; -X- is -L1 -CH2CH3-, wherein L1 is an immunoglobulin lgG1 hinge having the amino acid sequence comprising any one of SEQ ID NOs:120-192 and wherein -CH2CH3- in certain embodiments is a human lgG1 CH2CH3 region or a variant thereof lacking one or more effector functions; L2 is a linker peptide having an amino acid sequence comprising any one of SEQ ID NOS:193-360 and 745-748; and BD2 is a binding domain that binds slL6xR or a target molecule other than slL6xR. In certain

embodiments, BD2 may be a receptor ectodomain. In certain embodiments, the binding polypeptide may comprise the format N-BD1 -X-L2-LIG-C where LIG is a ligand for a receptor {e.g., where the ligand may be a cytokine).

Yet another aspect of the present disclosure provides a fusion

polypeptide comprising the following structure: N-BD1 -X-L2-BD2-C wherein: BD1 comprises a binding domain that specifically binds slL6xR or a target molecule other than slL6xR; -X- is -L1 -CH2CH3-, wherein L1 is an

immunoglobulin lgG1 hinge region having the amino acid sequence comprising any one of SEQ ID NOs:120-192 and wherein -CH2CH3- comprises human lgG1 CH2 and CH3 domains or a variant thereof lacking one or more effector functions; L2 is a linker peptide having an amino acid sequence comprising any one of SEQ ID NOS:193-360 and 745-748; and BD2 is a binding domain that specifically binds slL6xR or a target molecule other than slL6xR. In one embodiment of the fusion polypeptide, BD1 is a TNF antagonist or a TGF antagonist and BD2 is a binding domain that specifically binds a human slL6xR complex with higher affinity than a binding domain comprising the VH and VL amino acid sequences as set forth in SEQ ID NOs:3 and 4, respectively, or than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2. In a further embodiment of the fusion polypeptide, BD2 comprises: (a) a VL region comprising: a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ; or (b) a VH region comprising: a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or (c) a VL of (a) and a VH of (b). In certain other embodiments, the polypeptide of the present disclosure also include polypeptide heterodimers formed between two different single chain polypeptides comprising a binding domain, via natural heterodimerization of an immunoglobulin CH1 region and an immunoglobulin light chain constant region (CL), such as those described in published PCT applications

WO201 1 /090762 and WO201 1/090754.

A "polypeptide heterodimer," "heterodimer," or "Interceptor," as used herein, refers to a dimer formed from two different single chain binding polypeptides (also referred to as single chain fusion polypeptides). In certain embodiments, a polypeptide heterodimer comprises at least one chain longer (long chain) than the other (short chain). This term does not include an antibody formed from four single chain polypeptides (i.e., two light chains and two heavy chains). A "dimer" refers to a biological entity that consists of two subunits associated with each other via one or more forms of intramolecular forces, including covalent bonds (e.g., disulfide bonds) and other interactions (e.g., electrostatic interactions, salt bridges, hydrogen bonding, and

hydrophobic interactions), and is stable under appropriate conditions (e.g., under physiological conditions, in an aqueous solution suitable for expressing, purifying, and/or storing recombinant proteins, or under conditions for non- denaturing and/or non-reducing electrophoresis).

A "single chain polypeptide", "single chain binding polypeptide" or a "single chain fusion polypeptide" is a single, linear and contiguous arrangement of covalently linked amino acids. It does not include two polypeptide chains that link together in a non-linear fashion, such as via an interchain disulfide bond (e.g., a half immunoglobulin molecule in which a light chain links with a heavy chain via a disulfide bond). In certain embodiments, a single chain polypeptide may have or form one or more intrachain disulfide bonds. A single chain polypeptide may or may not have a binding domain as described above. For example, in certain embodiments, two single chain polypeptides are constructed such that they form a heterodimer wherein one single chain polypeptide member of the heterodimer pair contains a binding domain and the other member of the pair does not. Thus, in this embodiment, the heterodimer formed functions as a binding molecule by function of the binding domain in one of the heterodimer member polypeptide chains.

An "immunoglobulin heterodimerization domain," as used herein, refers to an immunoglobulin domain ("first immunoglobulin heterodimerization domain") that preferentially interacts or associates with a different

immunoglobulin domain ("second immunoglobulin heterodimerization domain") wherein the interaction of the different heterodimerization domains substantially contributes to or efficiently promotes heterodimerization (i.e., the formation of a dimer between two different polypeptides, which is also referred to as a heterodimer). Representative immunoglobulin heterodimerization domains of the present disclosure include an immunoglobulin CH1 region, an

immunoglobulin CL region [e.g., CK or CA isotypes), or derivatives thereof, as provided herein.

In certain embodiments, a binding polypeptide of the invention may comprise a dimerization or heterodimerization domain. A "binding polypeptide heterodimer" or "heterodimer," as used herein, refers to a dimer formed from two different single chain binding polypeptides.

Dimerization/heterodimerization domains may be used where it is desired to form homo or heterodimers from two single chain binding

polypeptides, where one or both single chain polypeptides comprise one or more binding domains. It should be noted that in certain embodiments, one single chain polypeptide member of certain heterodimers described herein may not contain a binding domain. See, e.g., Table 4 of published application WO201 1/090761 . These single chain polypeptide members lacking a binding domain may contain any of the components of binding polypeptides as described herein [e.g., Fc regions, hinges, linkers,

dimerization/heterodimerization domains, junctional amino acids, etc.).

However, when formed in dimer/heterodimer, the complex contains at least one binding domain. In certain embodiments, the binding polypeptides comprise a

"dimerization domain," which refers to an amino acid sequence that is capable of promoting the association of at least two single chain polypeptides or proteins via non-covalent or covalent interactions, such as by hydrogen bonding, electrostatic interactions, salt bridges, Van der Waal's forces, disulfide bonds, hydrophobic interactions, or the like, or any combination thereof.

Exemplary dimerization domains include immunoglobulin heavy chain constant regions. It should be understood that a dimerization domain can promote the formation of dimers or higher order multimer complexes (such as trimers, tetramers, pentamers, hexamers, septamers, octamers, etc.).

In certain embodiments of the fusion polypeptide or the isolated polypeptides described herein, the fusion polypeptide or the isolated

polypeptide is contained in a first single chain polypeptide comprising a first heterodimerization domain that is capable of associating with a second single chain polypeptide comprising a second heterodimerization domain that is not the same as the first heterodimerization domain, wherein the associated first and second single chain polypeptides form a polypeptide heterodimer.

Where heterodimerization is desired, the heterodimerization domains of a binding polypeptide heterodimer are different from each other and thus may be differentially modified to facilitate heterodimerization of both chains and to minimize homodimerization of either chain. Heterodimerization domains provided herein allow for efficient heterodimerization between different polypeptides and facilitate purification of the resulting binding polypeptide heterodimers.

As provided herein, heterodimerization domains useful for promoting heterodimerization of two different single chain polypeptides {e.g., one short and one long) according to the present disclosure include immunoglobulin CH1 and CL domains, for instance, human CH1 and CL domains. In certain embodiments, an immunoglobulin heterodimerization domain is a wild type CH1 region, such as a wild type lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2 IgD, IgE, or IgM CH1 region. In further embodiments, an immunoglobulin heterodimerization domain is a wild type human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM CH1 region as set forth in SEQ ID NOS:363-371 , respectively. In certain embodiments, an immunoglobulin heterodimerization domain is a wild type human lgG1 CH1 region as set forth in SEQ ID NO:82, which may, in certain embodiments, be used in a construct herein without the terminal "RT" residues.

In further embodiments, an immunoglobulin heterodimerization domain is an altered immunoglobulin CH1 region, such as an altered lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2 IgD, IgE, or IgM CH1 region. In certain embodiments, an immunoglobulin heterodimerization domain is an altered human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM CH1 region. In still further

embodiments, a cysteine residue of a wild type CH1 region {e.g., a human CH1 ) involved in forming a disulfide bond with a wild type immunoglobulin CL domain {e.g., a human CL) is deleted or substituted in the altered

immunoglobulin CH1 region such that a disulfide bond is not formed between the altered CH1 region and the wild type CL domain.

In certain embodiments, an immunoglobulin heterodimerization domain is a wild type CL domain, such as a wild type CK domain or a wild type CA domain. In particular embodiments, an immunoglobulin heterodimerization domain is a wild type human CK or human CA domain as set forth in SEQ ID NOS:372 and 373, respectively. In further embodiments, an immunoglobulin heterodimerization domain is an altered immunoglobulin CL domain, such as an altered CK or CA domain, for instance, an altered human CK or human CA domain.

In certain embodiments, a cysteine residue of a wild type CL domain {e.g., a human CL) involved in forming a disulfide bond with a wild type immunoglobulin CH1 region {e.g., a human CH1 ) is deleted or substituted in the altered immunoglobulin CL domain. Such altered CL domains may further comprise an amino acid deletion at their amino termini. An exemplary CK domain is set forth in SEQ ID NO:374, in which the first arginine and the last cysteine of the wild type human Ck domain are both deleted. An exemplary CA domain is set forth in SEQ ID NO:375, in which the first arginine of a wild type human CA domain is deleted and the cysteine involved in forming a disulfide bond with a cysteine in a CH1 region is substituted by a serine.

In further embodiments, an immunoglobulin heterodimerization domain is an altered CK domain that contains one or more amino acid substitutions, as compared to a wild type CK domain, at positions that may be involved in forming the interchain-hydrogen bond network at a CK-CK interface. For example, in certain embodiments, an immunoglobulin heterodimerization domain is an altered human CK domain having one or more amino acids at positions N29, N30, Q52, V55, T56, S68 or T70 that are substituted with a different amino acid. The numbering of the amino acids is based on their positions in the altered human CK sequence as set forth in SEQ ID NO:374. In certain embodiments, an immunoglobulin heterodimerization domain is an altered human CK domain having one, two, three or four amino acid

substitutions at positions N29, N30, V55, or T70. The amino acid used as a substitute at the above-noted positions may be an alanine, or an amino acid residue with a bulk side chain moiety such as arginine, tryptophan, tyrosine, glutamate, glutamine, or lysine. Exemplary altered human CK domains are set forth in SEQ ID NOS: 376-412. Examples of altered human Ck domains are provided in SEQ ID NOS:413 and 414 in which amino acid residues 30, 55 and 70 have been modified. These two Ck variants are referred to as Ck (YAE) and Ck (EAE), respectively, referring to the three replacement residues. Certain altered human CK domains can facilitate heterodimerization with a CH1 region, but minimize homodimerization with another CK domain. Representative altered human CK domains are set forth in SEQ ID NOS:415 (N29W V55A T70A), 416 (N29Y V55A T70A), 417 (T70E N29A N30A V55A), 418 (N30R V55A T70A), 419 (N30K V55A T70A), 420 (N30E V55A T70A), 421 (V55R N29A N30A), 422 (N29W N30Y V55A T70E), 423 (N29Y N30Y V55A T70E), 414 (N30E V55A T70E), and 413 (N30Y V55A T70E).

In further embodiments, other altered human CK domains include N30D V55A T70E (DAE); N30M V55A T70E (MAE); N30S V55A T70E (SAE); and N30F V55A T70E (FAE). In further embodiments, specific altered CH1 domains may be

appropriately paired with particular altered human CK domains to destabilize homodimerization. In this regard, illustrative altered domain pairs include CK L29E + CH1 V68K and CK L29K + CH1 V68E.

In certain embodiments, in addition to or alternative to the mutations in

Ck domains described herein, both the immunoglobulin heterodimerization domains (i.e., immunoglobulin CH1 and CL domains) of a binding polypeptide heterodimer have mutations so that the resulting immunoglobulin

heterodimerization domains form salt bridges (i.e., ionic interactions) between the amino acid residues at the mutated sites. For example, the immunoglobulin heterodimerization domains of a binding polypeptide heterodimer may be a mutated CH1 domain in combination with a mutated Ck domain. In the mutated CH1 domain, valine at position 68 (V68) of the wild type human CH1 domain is substituted by an amino acid residue having a negative charge (e.g., aspartate or glutamate), whereas leucine at position 29 (L29) of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted is substituted by an amino acid residue having a positive charge (e.g., lysine, arginine or histidine). The charge-charge interaction between the amino acid residue having a negative charge of the resulting mutated CH1 domain and the amino acid residue having a positive charge of the resulting mutated Ck domain forms a salt bridge, which stabilizes the heterodimeric interface between the mutated CH1 and Ck domains. Alternatively, V68 of the wild type CH1 may be substituted by an amino acid residue having a positive charge, whereas L29 of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted may be substituted by an amino acid residue having a negative charge. Exemplary mutated CH1 domains in which V68 is substituted by an amino acid with either a negative or positive charge include V68K and V68E substituted CH1 domains. Exemplary mutated CK domains in which L29 is substituted by an amino acid with either a negative or positive charge include L29E and L29K substituted CK domains. In certain embodiments, the terminal cysteine residue present in wild type CK is deleted. Positions other than V68 of human CH1 domain and L29 of human Ck domain may be substituted with amino acids having opposite charges to produce ionic interactions between the amino acids in addition or alternative to the mutations in V68 of CH1 domain and L29 of Ck domain. Such positions can be identified by any suitable method, including random mutagenesis, analysis of the crystal structure of the CH1 -Ck pair to identify amino acid residues at the CH1 -Ck interface, and further identifying suitable positions among the amino acid residues at the CH1 -Ck interface using a set of criteria {e.g., propensity to engage in ionic interactions, proximity to a potential partner residue, etc.).

In certain embodiments, where binding polypeptide heterodimers are desired, the single chain polypeptides used may contain only one pair of heterodimerization domains. For example, a first chain of a binding polypeptide heterodimer may comprise a CH1 region as a heterodimerization domain, while a second chain may comprise a CL domain {e.g., a CK or CA) as a

heterodimerization domain. Alternatively, a first chain may comprise a CL region {e.g., a CK or CA) as a heterodimerization domain, while a second chain may comprise a CH1 region as a heterodimerization domain. As set forth herein, the heterodimerization domains of the first and second chains are capable of associating to form a binding polypeptide heterodimer of this disclosure.

In certain other embodiments, binding polypeptides may have two pairs of heterodimerization domains. For example, a first chain of a binding polypeptide heterodimer may comprise two CH1 regions, while a second chain may have two CL domains that associate with the two CH1 regions in the first chain. Alternatively, a first chain may comprise two CL domains, while a second chain may have two CH1 regions that associate with the two CL domains in the first chain. In certain embodiments, a first chain polypeptide comprises a CH1 region and a CL domain, while a second chain polypeptide comprises a CL domain and a CH1 region that associate with the CH1 region and the CL domain, respectively, of the first chain polypeptide. In the embodiments where a binding polypeptide heterodimer comprises only one heterodimenzation pair (i.e., one heterodimenzation domain in each chain), the heterodimenzation domain of each chain may be located amino terminal to the Fc region portion of that chain. Alternatively, the

heterodimerization domain in each chain may be located carboxyl terminal to the Fc region portion of that chain.

In the embodiments where a binding polypeptide heterodimer comprises two heterodimerization pairs (i.e., two heterodimerization domains in each chain), both heterodimerization domains in each chain may be located amino terminal to the Fc region portion of that chain. Alternatively, both

heterodimerization domains in each chain may be located carboxyl terminal to the Fc region portion of that chain. In further embodiments, one

heterodimerization domain in each chain may be located amino terminal to the Fc region portion of that chain, while the other heterodimerization domain of each chain may be located carboxyl terminal to the Fc region portion of that chain. In other words, in those embodiments, the Fc region portion is interposed between the two heterodimerization domains of each chain.

In certain embodiments, a polypeptide heterodimer as described herein comprises (i) a single chain polypeptide ("first single chain polypeptide") having a first immunoglobulin heterodimerization domain and (ii) another single chain polypeptide ("second single chain polypeptide") having a second

heterodimerization domain that is not the same as the first heterodimerization domain, wherein the first and second heterodimerization domains substantially contribute to or efficiently promote formation of the polypeptide heterodimer. The interaction(s) between the first and second heterodimerization domains substantially contributes to or efficiently promotes the heterodimerization of the first and second single chain polypeptides if there is a statistically significant reduction in the dimerization between the first and second single chain polypeptides in the absence of the first heterodimerization domain and/or the second heterodimerization domain. In certain embodiments, when the first and second single chain polypeptides are co-expressed, at least about 60%, at least about 60% to about 70%, at least about 70% to about 80%, at least about 80% to about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and at least about 90% to about 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second single chain polypeptides form heterodimers with each other.

The heterodimerization technology described herein has one or more of the following advantages: (1 ) minimal immunogenicity of the polypeptide heterodimers because the dimers are formed via natural heterodimerization of an immunoglobulin CH1 region and an immunoglobulin CL region; (2) efficient production and purification of polypeptide heterodimers of the present disclosure is possible by co-expressing the two different single chain

polypeptides, as shown in the examples; (3) the ability to mediate Fc effector functions {e.g., CDC, ADCC, ADCP), which can be modulated up or down by mutagenesis, and a longer serum half-life because each chain of a polypeptide heterodimer according to the present disclosure has an Fc region portion {e.g., immunoglobulin CH2 and CH3 domains); and (4) polypeptide heterodimers of the present disclosure having a size that is typically smaller than an antibody molecule, which can allow for better tissue penetration, such as into a solid malignancy.

In one aspect, the present disclosure provides a heterodimer that comprises only a single binding domain, i.e., a slL6xR binding domain. The heterodimer is comprised of a longer single chain polypeptide (which has a slL6xR binding domain) and a shorter single chain polypeptide (which does not have any binding domain). In addition, both chains of the heterodimer further each comprise an Fc region portion {e.g., immunoglobulin CH2 and/or CH3 domains).

More particularly, the present disclosure provides single chain binding polypeptides and polypeptide heterodimers thereof that contain a single slL6xR binding domain and have heterodimerization domain pairs of CK-CH1 or CA-CH1 , or a combination of these pairs. In the simplest form, polypeptide heterodimers (also referred to as Interceptors) are made by co-expressing two unequal chains, one chain having a CK or CA domain and the other chain having a CH1 region. For example, the first single chain binding polypeptide, designated the long chain, has a slL6xR binding domain in the form of scFv and a CH1 heterodimerization domain, whereas the other chain, designated the short chain, lacks a binding domain but has a CK heterodimerization domain. Polypeptide heterodimers (Interceptors) will generally bind monovalently to the slL6xR target protein and are ideal for blocking receptor/I igand or

receptor/receptor interactions and preventing cell activation through receptor cross-linking. Other various advantages over, for example, a Fab, include a longer serum half-life and ease of purification due to the presence of the Fc domains. The interceptors may have a slL6xR binding domain at the amino terminus or at the carboxyl terminus.

In another aspect, the present disclosure provides a polypeptide heterodimer ("multi-specific heterodimer") formed by the association of two different single chain polypeptides wherein there is more than one binding domain, in particular at least one slL6xR binding domain and at least one binding domain that binds a target other than slL6xR. In certain embodiments, a heterodimer may be bispecific or may be multispecific. In this aspect, the present disclosure provides a polypeptide heterodimer wherein the first single chain binding polypeptide (SCP-I) comprises, consists essentially of, or consists of from one to four binding domains that specifically bind from one to four targets, a hinge (H-l), an immunoglobulin heterodimerization domain (HD-I), and an Fc region portion (FRP-I), whereas the second single chain polypeptide (SCP-II) comprises, consists essentially of, or consists of from zero to four binding domains that specifically bind from zero to four targets, a hinge (H-l I), an immunoglobulin heterodimerization domain (HD-II), and an Fc region portion (FRP-II), provided that the polypeptide heterodimer comprises at least two binding domains that specifically bind to at least two different targets. The H-l and H-ll may have the same sequence, but may be different. The FRP-I and FRP-II may have the same sequence, but may be different. The individual components of the polypeptide heterodimers of the present disclosure are described in detail herein.

If a single chain polypeptide of a multi-specific heterodimer comprises a single binding domain, the binding domain may be located either amino or carboxyl terminal to the Fc region portion of the single chain polypeptide. For example, a single chain polypeptide comprising two binding domains may have one binding domain located amino terminal and the other carboxyl terminal to the Fc region portion of the single chain polypeptide, or both binding domains may be amino terminal or both carboxyl terminal to the Fc region portion. In another example, a single chain polypeptide may comprise three binding domains wherein (a) two binding domains are amino terminal on different single chain proteins and the third binding domain is carboxyl terminal to the Fc region portion on either SCP-I or SCP-II, (b) two binding domains are carboxyl terminal on different single chain proteins and the third binding domain is amino terminal to the Fc region portion on either SCP-I or SCP-II. In still a further example, a polypeptide heterodimer may comprise four binding domains, wherein two binding domains are located amino terminal to the Fc region portion on different single chain proteins and the other two binding domains are located carboxyl terminal to the Fc region portion on different chains. Alternatively, in any of these embodiments, two binding domains may be linked to each other in tandem and located on either SCP-I or SCP-II or both, depending on the number of binding domains present - the tandem stacking is used when five to eight binding domains combined are present in SCP-I and SCP-II.

Thus, in certain embodiments, a heterodimer comprises at least one slL6xR binding domain and may comprise one or more additional binding domains that bind to a heterologous target protein such as, but not limited to, TNFa, TGF , or any other heterologous target protein, e.g., as described herein. In one particular embodiment, the first single chain polypeptide comprises an antislL6xR binding domain and the second single chain

polypeptide comprises a TNFa binding domain. In an additional embodiment, the first single chain polypeptide comprises a slL6xR binding domain and the second single chain polypeptide comprises a TNFR ectodomain.

In a related aspect, the present disclosure provides a polypeptide heterodimer formed by the association of two different single chain polypeptides that comprise two or more binding domains, each of which binds slL6xR. Such a polypeptide heterodimer may be similar to a multi-specific heterodimer described herein except that its binding domains bind only to slL6xR as opposed to the binding domains of the multi-specific heterodimer that bind at least two different targets.

To efficiently produce any of the polypeptides comprising binding domains described herein, a leader peptide may be used to facilitate secretion of expressed polypeptides. Using any of the conventional leader peptides (signal sequences) is expected to direct nascently expressed polypeptides into a secretory pathway and to result in cleavage of the leader peptide from the mature polypeptide at or near the junction between the leader peptide and the polypeptide. A particular leader peptide will be chosen based on considerations known in the art, such as using sequences encoded by polynucleotides that allow the easy inclusion of restriction endonuclease cleavage sites at the beginning or end of the coding sequence for the leader peptide to facilitate molecular engineering, provided that such introduced sequences specify amino acids that either do not interfere unacceptably with any desired processing of the leader peptide from the nascently expressed protein or do not interfere unacceptably with any desired function of a polypeptide if the leader peptide is not cleaved during maturation of the polypeptides. Exemplary leader peptides of this disclosure include natural leader sequences (i.e., those expressed with the native protein) or use of heterologous leader sequences, such as

H₃N-MDFQVQIFSFLLISASVIMSRG(X)n-CO₂H, wherein X is any amino acid and n is zero to three (SEQ ID NOS:740-743) or

H3N-MEAPAQLLFLLLLWLPDTTG-CO2H (SEQ ID NO:744).

As noted herein, variants and derivatives of binding domains, such as ectodomains, light and heavy variable regions, and CDRs described herein, are contemplated. In one example, insertion variants are provided wherein one or more amino acid residues supplement a specific binding agent amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the specific binding agent amino acid sequence. Variant products of this disclosure also include mature specific binding agent products, i.e., specific binding agent products wherein a leader or signal sequence is removed, and the resulting protein having additional amino terminal residues. The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from a specific protein. Polypeptides with an additional methionine residue at position -1 are contemplated, as are polypeptides of this disclosure with additional methionine and lysine residues at positions -2 and -1 . Variants having additional Met, Met-Lys, or Lys residues (or one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.

As used herein, "amino acids" refer to a natural (those occurring in nature) amino acid, a substituted natural amino acid, a non-natural amino acid, a substituted non-natural amino acid, or any combination thereof. The designations for natural amino acids are herein set forth as either the standard one- or three-letter code. Natural polar amino acids include asparagine (Asp or N) and glutamine (Gin or Q); as well as basic amino acids such as arginine (Arg or R), lysine (Lys or K), histidine (His or H), and derivatives thereof; and acidic amino acids such as aspartic acid (Asp or D) and glutamic acid (Glu or E), and derivatives thereof. Natural hydrophobic amino acids include tryptophan (Trp or W), phenylalanine (Phe or F), isoleucine (lie or I), leucine (Leu or L), methionine (Met or M), valine (Val or V), and derivatives thereof; as well as other non-polar amino acids such as glycine (Gly or G), alanine (Ala or A), proline (Pro or P), and derivatives thereof. Natural amino acids of intermediate polarity include serine (Ser or S), threonine (Thr or T), tyrosine (Tyr or Y), cysteine (Cys or C), and derivatives thereof. Unless specified otherwise, any amino acid described herein may be in either the D- or L-configuration. Substitution variants include those polypeptides wherein one or more amino acid residues in an amino acid sequence are removed and replaced with alternative residues. In some embodiments, the substitutions are conservative in nature; however, this disclosure embraces substitutions that are also non- conservative. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 1 (see WO 97/09433, page 10, published March 13, 1997), immediately below.

Table 1 . Conservative Substitutions I

Alternatively, conservative amino acids can be grouped as described in Lehninger (Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71 -77) as set out in Table 2, immediately below.

Table 2. Conservative Substitutions II

Variants or derivatives can also have additional amino acid residues which arise from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired

polypeptide having an additional glycine residue at position -1 after cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated, including those wherein histidine tags are incorporated into the amino acid sequence, generally at the carboxyl and/or amino terminus of the sequence.

Deletion variants are also contemplated wherein one or more amino acid residues in a binding domain of this disclosure are removed. Deletions can be effected at one or both termini of the fusion protein, or from removal of one or more residues within the amino acid sequence.

In certain illustrative embodiments, binding polypeptides of the invention are glycosylated, the pattern of glycosylation being dependent upon a variety of factors including the host cell in which the protein is expressed (if prepared in recombinant host cells) and the culture conditions. This disclosure also provides derivatives of binding polypeptides. In certain embodiments, the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties. Derivatives of this disclosure may be prepared to increase circulating half-life of a specific binding domain polypeptide, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs.

This disclosure further embraces binding polypeptides that are covalently modified or derivatized to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or

polypropylene glycol, as described U .S. Patent NOs: 4,640,835; 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192 and 4,179,337. Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, and other carbohydrate-based polymers, poly-(N-vinyl pyrrolidone)- polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers. Some embodiments utilize polyethylene glycol (PEG)-derivatized proteins. Water-soluble polymers may be bonded at specific positions, for example at the amino terminus of the proteins and polypeptides according to this disclosure, or randomly attached to one or more side chains of the polypeptide. The use of PEG for improving therapeutic capacities is described in US Patent No. 6,133,426.

In one embodiment, the binding polypeptide is a fusion protein that comprises an immunoglobulin or an Fc fusion protein. Such a fusion protein can have a long half-life, e.g., several hours, a day or more, or even a week or more, especially if the Fc domain is capable of interacting with FcRn, the neonatal Fc receptor. The binding site for FcRn in an Fc domain is also the site at which the bacterial proteins A and G bind. The tight binding between these proteins can be used as a means to purify antibodies or fusion proteins of this disclosure by, for example, employing protein A or protein G affinity

chromatography during protein purification. In certain embodiments, the Fc domain of the fusion protein is optionally mutated to eliminate interaction with FcyRI-l 11 while retaining FcRn interaction.

Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the

polypeptide and non-polypeptide fractions. Further purification using

chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) is frequently desired. Analytical methods particularly suited to the preparation of a pure polypeptide are ion- exchange chromatography; exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Particularly efficient methods of purifying peptides are fast protein liquid chromatography and HPLC.

Certain aspects of the present disclosure concern the purification, and in particular embodiments, the substantial purification, of a polypeptide. The terms "purified polypeptide" and "purified fusion protein" are used

interchangeably herein and refer to a composition, isolatable from other components and that is purified to any degree relative to its naturally obtainable state. A purified polypeptide therefore also refers to a polypeptide, free from the envislL6xRment in which it may naturally occur.

Generally, "purified" will refer to a polypeptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation refers to a polypeptide composition in which the polypeptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or more of the polypeptide, by weight, in the composition.

Various methods for quantifying the degree of purification are known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific binding activity of an active fraction, or assessing the amount of polypeptide in a fraction by SDS/PAGE analysis. One method for assessing the purity of a protein fraction is to calculate the binding activity of the fraction, to compare it to the binding activity of the initial extract, and to thus calculate the degree of purification, herein assessed by a "-fold purification number." The actual units used to represent the amount of binding activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed fusion protein exhibits a detectable binding activity.

Various techniques suitable for use in protein purification are well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like, or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, and affinity chromatography;

isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein.

There is no general requirement that the binding polypeptide always be provided in its most purified state. Indeed, it is contemplated that less substantially purified proteins will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in greater purification than the same technique utilizing a low pressure

chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining binding activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al. (1977) Biochem. Biophys. Res. Comm. 76:425). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified fusion protein expression products may vary.

This disclosure provides polynucleotides (isolated or purified or pure polynucleotides) encoding the binding polypeptides as described herein, vectors (including cloning vectors and expression vectors) comprising such polynucleotides, and cells {e.g., host cells) transformed or transfected with a polynucleotide or vector according to this disclosure.

In certain embodiments, a polynucleotide (DNA or RNA) encoding a binding domain of this disclosure, or polypeptides containing one or more such binding domains is contemplated. Expression cassettes encoding fusion protein constructs are provided in the examples and the sequence listing appended hereto.

The present disclosure also relates to vectors that include a

polynucleotide of this disclosure and, in particular, to recombinant expression constructs. In one embodiment, this disclosure contemplates a vector comprising a polynucleotide encoding a slL6xR binding domain or other binding domain and polypeptides thereof, along with other polynucleotide sequences that cause or facilitate transcription, translation, and processing of such protein- encoding sequences.

Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, in Sambrook et ai, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989). Exemplary cloning/expression vectors include cloning vectors, shuttle vectors, and expression constructs, that may be based on plasmids, phagemids, phasmids, cosmids, viruses, artificial chromosomes, or any nucleic acid vehicle known in the art suitable for amplification, transfer, and/or expression of a polynucleotide contained therein.

As used herein, "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Exemplary vectors include plasmids, yeast artificial chromosomes, and viral genomes. Certain vectors can autonomously replicate in a host cell, while other vectors can be integrated into the genome of a host cell and thereby are replicated with the host genome. In addition, certain vectors are referred to herein as

"recombinant expression vectors" (or simply, "expression vectors"), which contain nucleic acid sequences that are operatively linked to an expression control sequence and, therefore, are capable of directing the expression of those sequences.

In certain embodiments, expression constructs are derived from plasmid vectors. Illustrative constructs include modified pNASS vector (Clontech, Palo Alto, CA), which has nucleic acid sequences encoding an ampicillin resistance gene, a polyadenylation signal and a T7 promoter site; pDEF38 and pNEF38 (CMC ICOS Biologies, Inc.), which have a CHEF1 promoter; and pD18 (Lonza), which has a CMV promoter. Other suitable mammalian expression vectors are well known (see, e.g., Ausubel et al., 1995; Sambrook et al., supra; see also, e.g., catalogs from Invitrogen, San Diego, CA; Novagen, Madison, Wl;

Pharmacia, Piscataway, NJ). Useful constructs may be prepared that include a dihydrofolate reductase (DHFR)-encoding sequence under suitable regulatory control, for promoting enhanced production levels of the fusion proteins, which levels result from gene amplification following application of an appropriate selection agent [e.g., methotrexate).

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, as described above. A vector in operable linkage with a polynucleotide according to this disclosure yields a cloning or expression construct. Exemplary cloning/expression constructs contain at least one expression control element, e.g., a promoter, operably linked to a polynucleotide of this disclosure. Additional expression control elements, such as enhancers, factor-specific binding sites, terminators, and ribosome binding sites are also contemplated in the vectors and cloning/expression constructs according to this disclosure. The heterologous structural sequence of the polynucleotide according to this disclosure is assembled in appropriate phase with translation initiation and termination sequences. Thus, for example, the protein-encoding nucleic acids as provided herein may be included in any one of a variety of expression vector constructs as a recombinant expression construct for expressing such a protein in a host cell.

The appropriate DNA sequence(s) may be inserted into a vector, for example, by a variety of procedures. In general, a DNA sequence is inserted into an appropriate restriction endonuclease cleavage site(s) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA

polymerase, restriction endonucleases and the like, and various separation techniques are contemplated. A number of standard techniques are described, for example, in Ausubel et al. (Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, MA, 1993); Sambrook et al. (Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, NY, 1989); Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY, 1982); Glover (Ed.) (DNA Cloning Vol. I and II, IRL Press, Oxford, UK, 1985); Hames and Higgins (Eds.) (Nucleic Acid Hybridization, IRL Press, Oxford, UK, 1985); and elsewhere.

The DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequence (e.g., a constitutive promoter or a regulated promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include promoters of eukaryotic cells or their viruses, as described above. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is within the level of ordinary skill in the art, and preparation of recombinant expression constructs comprising at least one promoter or regulated promoter operably linked to a nucleic acid encoding a protein or polypeptide according to this disclosure is described herein. Variants of the polynucleotides of this disclosure are also contemplated. Variant polynucleotides are at least about 80 %, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to one of the

polynucleotides of defined sequence as described herein, or that hybridizes to one of those polynucleotides of defined sequence under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. The polynucleotide variants retain the capacity to encode a binding domain or fusion protein thereof having the functionality described herein.

The term "stringent" is used to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at about 65- 68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C (see Sambrook et ai, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used; however, the rate of hybridization will be affected. In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6x SSC, 0.05% sodium pyrophosphate at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides).

A further aspect of this disclosure provides a host cell transformed or transfected with, or otherwise containing, any of the polynucleotides or vector/expression constructs of this disclosure. The polynucleotides or cloning/expression constructs of this disclosure are introduced into suitable cells using any method known in the art, including transformation, transfection and transduction. Host cells include the cells of a subject undergoing ex vivo cell therapy including, for example, ex vivo gene therapy. Eukaryotic host cells contemplated as an aspect of this disclosure when harboring a polynucleotide, vector, or protein according to this disclosure include, in addition to a subject's own cells {e.g., a human patient's own cells), VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines (including modified CHO cells capable of modifying the glycosylation pattern of expressed multivalent binding molecules, see US Patent Application Publication No. 2003/01 15614), COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, Spodoptera frugiperda cells (e.g., Sf9 cells), Saccharomyces cerevisiae cells, and any other eukaryotic cell known in the art to be useful in expressing, and optionally isolating, a protein or peptide according to this disclosure. Also contemplated are prokaryotic cells, including Escherichia coli, Bacillus subtilis, Salmonella typhimurium, a Streptomycete, or any prokaryotic cell known in the art to be suitable for expressing, and optionally isolating, a protein or peptide according to this disclosure. In isolating protein or peptide from prokaryotic cells, in particular, it is contemplated that techniques known in the art for extracting protein from inclusion bodies may be used. The selection of an appropriate host is within the scope of those skilled in the art from the teachings herein. Host cells that glycosylate the fusion proteins of this disclosure are contemplated.

The term "recombinant host cell" (or simply "host cell") refers to a cell containing a recombinant expression vector. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

Recombinant host cells can be cultured in a conventional nutrient medium modified as appropriate for activating promoters, selecting

transformants, or amplifying particular genes. The culture conditions for particular host cells selected for expression, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan. Various

mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman (1981 ) Cell 23:175, and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and, optionally, enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5'-flanking nontranscribed sequences, for example, as described herein regarding the preparation of multivalent binding protein expression constructs. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including calcium phosphate transfection, DEAE-Dextran-mediated transfection, or electroporation (Davis et al. (1986) Basic Methods in Molecular Biology).

In one embodiment, a host cell is transduced by a recombinant viral construct directing the expression of a protein or polypeptide according to this disclosure. The transduced host cell produces viral particles containing expressed protein or polypeptide derived from portions of a host cell membrane incorporated by the viral particles during viral budding. The present disclosure further provides for compositions comprising any of the polypeptides comprising binding domains as described herein. The polypeptides of the invention are slL6xR binding polypeptides.

The present disclosure also provides pharmaceutical compositions and unit dose forms that comprise any format of the binding polypeptides {e.g., anti- slL6xR antibody, single binding domain formats (SMIP, PIMS), multi-specific binding polypeptide formats {e.g., Xceptor), homodimeric and heterodimeric (Interceptor) as well as methods for using the compositions comprising any format of the slL6xR binding polypeptides described herein.

Compositions of this disclosure generally comprise a polypeptide comprising a binding domain and are polypeptides in any format described herein in combination with a pharmaceutically acceptable excipient, including pharmaceutically acceptable carriers and diluents. Pharmaceutical acceptable excipients will be nontoxic to recipients at the dosages and concentrations employed. They are well known in the pharmaceutical art and described, for example, in Rowe et al., Handbook of Pharmaceutical Excipients: A

Comprehensive Guide to Uses, Properties, and Safety, 5^th Ed., 2006.

Pharmaceutically acceptable carriers for therapeutic use are also well known in the pharmaceutical art, and are described, for example, in

Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro (Ed.) 1985). Exemplary pharmaceutically acceptable carriers include sterile saline and phosphate buffered saline at physiological pH. Preservatives, stabilizers, dyes and the like may be provided in the pharmaceutical

composition. In addition, antioxidants and suspending agents may also be used.

Pharmaceutical compositions may also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates (e.g., glucose, sucrose, dextrins), chelating agents {e.g., EDTA), glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary diluents. In one embodiment, the product is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.

The present disclosure also provides a method for treating a disease or disorder associated with, for example, excessive receptor-mediated signal transduction, comprising administering to a patient in need thereof a

therapeutically effective amount of any of the slL6xR binding proteins described herein. Increased production of IL-6, and thus IL-6 signaling, has been implicated in various disease processes, including Alzheimer's disease, autoimmunity {e.g., rheumatoid arthritis, SLE, psoriasis, colitis), inflammation, myocardial infarction, Paget's disease, osteoporosis, solid tumors (e.g., colon cancer, RCC prostatic and bladder cancers), certain neurological cancers, B- cell malignancies (e.g., Castleman's disease, some lymphoma subtypes, chronic lymphocytic leukemia, and, in particular, malignant melanoma). In some instances, IL-6 is implicated in proliferation pathways because it acts with other factors, such as heparin-binding epithelial growth factor and hepatocyte growth factor (see, e.g., Grant et al. (2002) Oncogene 21 :460; Badache and Hynes (2001 ) Cancer Res. 61 :383; Wang et al. (2002) Oncogene 21 :2584). Similarly, the TNF superfamily is known to be involved in a variety of disorders, such as cancer (tumorigenesis, including proliferation, migration, metastasis), autoimmunity (SLE, diabetes), chronic heart failure, bone resorption, and atherosclerosis, to name a few (see, e.g., Aggarwal (2003) Nature Rev. 3:745; Lin et al. (2008) Clin. Immunol. 126:13). Thus, some embodiments provide methods of treating one or more diseases, the method comprising

administering a polypeptide of the disclosure.

Exemplary diseases or disorders associated with excess receptor- mediated signal transduction include cancer (e.g., solid malignancy and hematologic malignancy) and a variety of inflammatory disorders.

In one embodiment, the present disclosure provides a method for treating, reducing the severity of or preventing inflammation or an inflammatory disease (see e.g., Camp et al. Ann. Surg. Oncol. 12:273-281 (2005); Correll, P.H. et al., Genes Funct. 1997 Feb;1 (1 ):69-83). For example, one embodiment of the invention provides a method for the treatment of inflammation or an inflammatory disease including, but not limited to Crohn's disease, colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), rheumatoid arthritis, asthma, systemic lupus erythematous, nephritis,

Parkinson's disease, ulcerative colitis, pre-term birth due to intra-amniotic inflammation, multiple sclerosis (MS), TNFRSFIA-associated periodic syndrome (TRAPS), Alzheimer's disease, arthritis, and various cardiovascular diseases such as, but not limited to, atherosclerosis and vasculitis. In certain embodiments, the inflammatory disease is selected from the group consisting of diabetes, gout, cryopyrin-associated periodic syndrome, and chronic obstructive pulmonary disorder. In this regard, one embodiment provides a method of treating, reducing the severity of or preventing inflammation or an inflammatory disease by administering to a patient in need thereof a therapeutically effective amount of a slL6xR binding protein as disclosed herein.

In one aspect, the present disclosure provides a method for inhibiting growth, metastasis or metastatic growth of a malignancy {e.g., a solid

malignancy or a hematologic malignancy), comprising administering to a patient in need thereof an effective amount slL6xR binding polypeptide of any format described herein or a composition thereof.

A wide variety of cancers, including solid malignancy and hematologic malignancy, are amenable to the compositions and methods disclosed herein. Types of cancer that may be treated include, but are not limited to, colon cancer; ovarian cancer; pancreatic cancer; prostate cancer, non-Hodgkin's lymphoma, kidney cancer, and lung cancer. Types of cancer that may be treated using the binding polypeptides and compositions thereof of the present disclosure include, but are not limited to adenocarcinoma of the breast, prostate, pancreas, colon and rectum; all forms of bronchogenic carcinoma of the lung (including squamous cell carcinoma, adenocarcinoma, small cell lung cancer and non-small cell lung cancer); myeloid; melanoma; hepatoma;

neuroblastoma; papilloma; apudoma; choristoma; branchioma; malignant carcinoid syndrome; carcinoid heart disease; and carcinoma {e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell). Additional types of cancers that may be treated include: histiocytic disorders; leukemia; histiocytosis malignant; Hodgkin's disease; immunoproliferative small; non- Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma;

myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma;

dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; trophoblastic tumor. Further, the following types of cancers are also contemplated as amenable to treatment: adenoma; cholangioma; cholesteatoma; cyclindroma; cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma; hepatoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma; myoblastoma;

myomma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma;

neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma; paraganglioma nonchromaffin; and glioblastoma multiforme. The types of cancers that may be treated also include, but are not limited to, angiokeratoma; angiolymphoid hyperplasia with eosinophilia; angioma sclerosing;

angiomatosis; glomangioma; hemangioendothelioma; hemangioma;

hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma;

cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasms;

nerofibromatosis; and cervical dysplasia.

Additional exemplary cancers that are also amenable to the

compositions and methods disclosed herein are B-cell cancers, including B-cell lymphomas [such as various forms of Hodgkin's disease, non-Hodgkins lymphoma (NHL) or central nervous system lymphomas], leukemias [such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia and chronic myoblastic leukemia] and myelomas (such as multiple myeloma). Additional B cell cancers include small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B- cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma/leukemia, B- cell proliferations of uncertain malignant potential, lymphomatoid

granulomatosis, and post-transplant lymphoproliferative disorder.

Any format of the binding polypeptides or compositions thereof of the present disclosure may be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection, or any combination thereof. In one embodiment, the slL6xR binding proteins or compositions thereof are administered parenterally. The term "parenteral," as used herein, includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site is contemplated as well. For instance, the invention includes methods of treating a patient comprising administering a therapeutically effective amount of the binding polypeptide of the invention or composition of the invention to a patient by intravenous injection.

The therapeutically effective dose depends on the type of disease, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the specific subject under consideration for treatment, concurrent medication, and other factors that those skilled in the medical arts will recognize. For example, an amount between 0.01 mg/kg and 1000 mg/kg (e.g., about 0.1 to 1 mg/kg, about 1 to 10 mg/kg, about 10-50 mg/kg, about 50-100 mg/kg, about 100-500 mg/kg, or about 500-1000 mg/kg) body weight (which can be administered as a single dose, daily, weekly, monthly, or at any appropriate interval) of active ingredient may be administered depending on the potency of a binding polypeptide of this disclosure.

Also contemplated is the administration of binding polypeptides or compositions thereof in combination with a second agent. A second agent may be one accepted in the art as a standard treatment for a particular disease state or disorder, such as in cancer or in an inflammatory disorder. Exemplary second agents contemplated include polyclonal antibodies, monoclonal antibodies, immunoglobulin-derived fusion proteins, chemotherapeutics, ionizing radiation, steroids, NSAIDs, anti-infective agents, or other active and ancillary agents, or any combination thereof.

A variety of other therapeutic agents may find use for administration with the binding polypeptides described herein. In one embodiment, the binding polypeptide is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, immune selective anti-inflammatory derivatives (imSAIDS), methotrexate, sulfasalazine, lefiunomide, anti-T F medications, cyclophosphamide and mycophenolate.

Second agents useful in combination with the binding protein or compositions thereof provided herein include anti-infective drugs, such as antibiotics antiviral and antifungal agents. Exemplary antibiotics include, for example, penicillin, cephalosporins, aminoglycosides, macrolides, quinolones and tetracyclines. Exemplary antiviral agents include, for example, reverse transcriptase inhibitors, protease inhibitors, antibodies, and interferons.

Exemplary antifungal agents include, for example, polyene antifungals {e.g., natamycin and rimocidin), imidazole, triazole, or thiazole antifungals {e.g., miconazone, ketoconazole, fluconazole, itraconazole, and abaungin), allylamines {e.g., terbinafine, naftifine), and echinocandins {e.g., anidulafungin and casposungin). In certain embodiments, a binding polypeptide and a second agent act synergistically. In other words, these two compounds interact such that the combined effect of the compounds is greater than the sum of the individual effects of each compound when administered alone (see, e.g., Berenbaum, Pharmacol. Rev. 41 :93, 1989).

In certain other embodiments, a binding polypeptide and a second agent act additively. In other words, these two compounds interact such that the combined effect of the compounds is the same as the sum of the individual effects of each compound when administered alone.

Second agents useful in combination with binding proteins or

compositions thereof provided herein may be steroids, NSAIDs, mTOR inhibitors {e.g., rapamycin (sirolimus), temsirolimus, deforolimus, everolimus, zotarolimus, curcumin, farnesylthiosalicylic acid), calcineurin inhibitors {e.g., cyclosporine, tacrolimus), anti-metabolites {e.g., mycophenolic acid,

mycophenolate mofetil), polyclonal antibodies {e.g., anti-thymocyte globulin), monoclonal antibodies {e.g., daclizumab, basiliximab, HERCEPTIN®

(trastuzumab), ERBITUX® (Cetuximab)), and CTLA4-lg fusion proteins {e.g., abatacept or belatacept). Illustrative second agents useful in combination with binding proteins or compositions thereof provided herein include, but are not limited to, infliximab, adalimumab, ocrelizumab, ofatumumab, golimumab, etanercept, certolizumab, ART621 , ATN-103, tocilizumab, CNTO-136, CNTO- 328, ALD-518, C326, CDP6038, REGN-88, CR5/18 and CP-690550.

Second agents useful for inhibiting growth of a solid malignancy, inhibiting metastasis or metastatic growth of a solid malignancy, or treating or ameliorating a hematologic malignancy include chemotherapeutic agents, ionizing radiation, and other anti-cancer drugs. Examples of chemotherapeutic agents contemplated as further therapeutic agents include alkylating agents, such as nitrogen mustards {e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil); bifunctional chemotherapeutics {e.g., bendamustine); nitrosoureas {e.g., carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU)); proteasome inhibitors {e.g. VELCADE® (bortezomib)); tyrosine kinase inhibitors (e.g. TARCEVA® (erlotinib) and TYKERB® (lapatinib)); ethyleneimines and methyl-melamines {e.g.,

triethylenemelamine (TEM), triethylene thiophosphoramide (thiotepa), and hexamethylmelannine (HMM, altretamine)); alkyl sulfonates {e.g., buslfan); and triazines {e.g., dacabazine (DTIC)); antimetabolites, such as folic acid analogues {e.g., methotrexate, trimetrexate, and pemetrexed (multi-targeted antifolate)); pyrimidine analogues (such as 5-fluorouracil (5-FU),

fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5- azacytidine, and 2,2'-difluorodeoxycytidine); and purine analogues (e.g., 6- mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate,

2-chlorodeoxyadenosine (cladribine, 2-CdA)); Type I topoisomerase inhibitors such as camptothecin (CPT), topotecan, and irinotecan; natural products, such as epipodophylotoxins {e.g., etoposide and teniposide); and vinca alkaloids {e.g., vinblastine, vincristine, and vinorelbine); anti-tumor antibiotics such as actinomycin D, doxorubicin, and bleomycin; radiosensitizers such as 5- bromodeozyuridine, 5-iododeoxyuridine, and bromodeoxycytidine; platinum coordination complexes such as cisplatin, carboplatin, and oxaliplatin;

substituted ureas, such as hydroxyurea; and methylhydrazine derivatives such as N-methylhydrazine (MIH) and procarbazine.

In certain embodiments, second agents useful for inhibiting growth metastasis or metastatic growth of a malignancy include multi-specific binding polypeptides or binding polypeptide heterodimers according to the present disclosure that bind to cancer cell targets other than slL6xR. In certain other embodiments, second agents useful for such treatments include polyclonal antibodies, monoclonal antibodies, and immunoglobulin-derived fusion proteins that bind to cancer cell targets.

Further therapeutic agents contemplated by this disclosure are referred to as immunosuppressive agents, which act to suppress or mask the immune system of the individual being treated. Immunosuppressive agents include, for example, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, glucocorticoids, disease-modifying antirheumatic drugs (DMARDs) for the treatment of arthritis, or biologic response modifiers. Compositions in the DMARD description are also useful in the treatment of many other autoimmune diseases aside from rheumatoid arthritis.

Exemplary NSAIDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib), and sialylates. Exemplary analgesics are chosen from the group consisting of acetaminophen, oxycodone, tramadol of

propoxyphene hydrochloride. Exemplary glucocorticoids are chosen from the group consisting of cortisone, dexamethasone, hydrocortisone,

methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers {e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists {e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab

(REMICADE®)), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.

It is contemplated the binding molecule composition and the second active agent may be given simultaneously in the same formulation.

Alternatively, the second agents may be administered in a separate formulation but concurrently {e.g., given within less than one hour of each other).

In certain embodiments, the second active agent may be administered prior to administration of a slL6xR binding polypeptide or a composition thereof. Prior administration refers to administration of the second active agent at least one hour prior to treatment with the slL6xR binding protein or the composition thereof. It is further contemplated that the active agent may be administered subsequent to administration of the binding molecule composition. Subsequent administration is meant to describe administration at least one hour after the administration of the binding molecule or the composition thereof.

This disclosure contemplates a dosage unit comprising a pharmaceutical composition of this disclosure. Such dosage units include, for example, a single-dose or a multi-dose vial or syringe, including a two-compartment vial or syringe, one comprising the pharmaceutical composition of this disclosure in lyophilized form and the other a diluent for reconstitution. A multi-dose dosage unit can also be, e.g., a bag or tube for connection to an intravenous infusion device.

As an additional aspect, the disclosure includes kits which comprise one or more compounds or compositions useful in the methods of this disclosure packaged in a manner which facilitates their use to practice methods of the disclosure. In a simplest embodiment, such a kit includes a compound or composition described herein as useful for practice of a method of the disclosure packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition to practice the method of the disclosure. In some embodiments, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition, e.g., according to a preferred or particular route of administration, or for practicing a screening assay. The kit may include a label that describes use of the binding molecule composition(s) in a method of the disclosure.

EXAMPLES

EXAMPLE 1

A2 ANTi HYPER IL-6 BINDING DOMAIN SMIP SPECIFICALLY BLOCKS HH IL6 INDUCED

TF-1 PROLIFERATION WITHOUT INHIBITING H I L-6 FUNCTION

A2 is an anti-human hyper IL-6 binding domain derived from a DYAX (Dyax, Cambridge, MA) phage display library as previously described in US Patent Publication No. 201 10142851 . As shown in Figure 1 , the A2 binding domain binds to human hyper IL-6 but does not bind to hlL-6. Blocking of IL6 or hyper IL6 (IL6xR; HIL6) induced cell proliferation of TF-I cells was examined for SMIP fusion proteins A2-SMIP #630 and #894 substantially as follows (see SEQ ID NOs:1 and 2 for the polynucleotide and amino acid sequences of the A2 SMIP binding molecule, respectively; the amino acid sequence of the VH and VL of the A2 binding domain are set forth in SEQ ID NOs: 3 and 4, respectively; the VH CDR1 , CDR2, and CDR3 sequences are set forth in SEQ ID NOs:5-7; the VL CDR1 , CDR2 and CDR3 sequences are set forth in SEQ ID NOs:8-10). The A2 SMIP is comprised of the A2 scFv binding domain having the (G4S)3 linker (see SEQ ID NO:33) between the variable domains, fused to a mutated lgG1 hinge ("SCC" hinge, i.e., the first cysteine residue is substituted with a serine residue) (see SEQ ID NO:34), fused to the lgG1 CH2CH3 null Fc region {e.g., Fc containing mutations to abrogate ADCC and CDC activity (see SEQ ID No:35).

Added to each well of a 96-well flat bottom plate were 0.3 x 10⁶ TF-I cells (human erythroleukemia cells that proliferate in response to both human IL-6 and hyper IL-6) in the fresh growth medium (10% FBS-RPMI 1640; 2mM L- glutamine; 100 units/ml penicillin; 100 ug/ml streptomycin; 10 mM HEPES; ImM sodium pyruvate; and 2ng/ml Hu GM-CSF) one day before use in proliferation assay. The cells were then harvested and washed twice with assay medium (same as growth medium except without GM-CSF, cytokine-free), then resuspended at 1 x 10⁵ cells/ml in assay medium. For blocking IL-6 activity, serial dilutions of an anti-hyper IL6 SMIP of interest or antibody was pre - incubated with a fixed concentration of recombinant human IL-6 (rhlL-6) (R&D Systems, Minneapolis, MN) or hyper IL-6 (HIL-6) in 96-well plates for 1 hour at 37°C, 5%CO₂. Controls used included anti-hlL-6 antibody (R&D Systems) and ENBREL®. After the pre-incubation period, 1 x10⁴ cells (in 100 μΙ) was added to each well. The final assay mixture, in a total volume of 200 μΙ/well, containing A2-SMIP, rhlL-6, or HIL-6 and cells was incubated at 37°C, 5%CO₂ for 72 hours. During the last 4-6 hours of culture, ³H-thymidine (20 Ci/ml in assay medium, 25 μΙ/well) was added. The cells were harvested onto UniFilter-96 GF/c plates and incorporated ³H-Thymidine was determined using TopCount reader (Packard). The data are presented as the Mean of cpm ± SD of triplicates. The percentage of blocking = 100 - (test cpm - control cpm

/maximum cpm- control cpm)^* 100.

As shown in Figure 2, the A2 SMIPs specifically block hHIL-6 induced TF-1 proliferation without inhibiting hlL-6 function.

EXAMPLE 2

AFFINITY MATURATION OF A2 ANTI HYPER IL-6 BINDING DOMAIN IDENTIFIES A2M1

BINDING DOMAIN WITH ENHANCED BINDING AFFINITY AND FUNCTIONAL BLOCKING OF

HYPER IL6 Site directed mutagenesis and phage display screening using hyper IL-6

(H-IL6) were carried out to identify affinity matured mutants of the A2 anti-hyper IL-6 binding domain.

The molecules screened by the Dyax system are usually in a Fab form. This is favorable when screening for antibodies, because the fusion molecule is expressed with separate VH and VLs coming together to form antibody-like fragments. For affinity maturation of the A2 binding domain, however, the Dyax machinery was utilized to present the A2 binding molecule in scFv form.

Reformatting the A2 binding domain into the phagemid pMID21 replaces the Fab and CH1 regions in the construct shown in Figure 3A with the A2 VHVL scFv. Figure 3B shows a diagram of the A2 VH-VL scFv construct and Figure 3C shows the sequence of the scFv heavy and light chain variable domains with the (G₄S)3 linker. The CDRs are highlighted and the framework regions are underlined. In particular, the VH and VL CDR3 were targeted for mutagenesis as described in further detail below.

The scFv domain of the anti-IL6/IL6R SMIP (ATH102) was amplified using VH and VL specific primers such that the resulting PCR product could be subcloned into a phagemid expression vector (pMID21 ) such that the anti- IL6/IL6R scFv domain could be expressed as a fusion protein with the bacteriophage gill protein. The anti-IL6/IL6R scFv phagemid expression vector was modified to incorporate mutations into each amino acid position of both the heavy and light chain CDR3 regions. The mutagenesis reaction was performed using

QuikChange Lightning Site-Directed Mutagenesis (Stratagene, now Agilent Technologies Inc., Santa Clara, CA). Mutagenic CDR3 primers were designed for each codon in such a way as to allow the first 2 nucleic acid positions to have any nucleic acid (denoted "N"). The third nucleic acid position was limited to thymine or guanine only (denoted "K"). Primers were designed to have NNK codons flanked by parental A2 sequence extending 15 nucleic acids both 5' and 3' of NNK. Full plasmid PCR was performed using the appropriate primers, using an annealing temperature of 65°C for 16 cycles. PCR product was digested with a CDR3-targeted restriction enzyme to eliminate unmodified parental plasmid. PCR product was transformed into E. coli TG1 via

electroporation and plated onto growth media containing appropriate antibiotic. Each of these VH-specific and VL-specific CDR3 mini-libraries was created and pooled separately. The mini-libraries were induced to produce phage displaying anti-IL6/IL6R scFv's with various CDR3 compositions.

Solution phage screening was performed by mixing mutant anti-IL6/IL6R scFv-displaying phage with biotinylated human hyper IL6. The resulting phage/protein complexes were then immobilized to streptavidin coated

DynaBeads (Invitrogen, Carlsbad, CA) and washed to remove weakly binding phage. The captured phage were recovered by infection of susceptible bacteria, amplified using standard phage growth conditions, and used for an additional round of screening. Three rounds of screenings were performed to produce the affinity selected VH CDR3 library. An affinity-selected VL CDR3 library was produced by the same method.

Phagemid DNA from the VH CDR3 screen was sequenced. Individual scFvs containing unique CDR3 mutations were converted to SMIP format for mammalian protein expression and quantitative binding assessment. Similarly, individual scFvs containing unique VL CDR3 mutations were converted to SMIP format for mammalian protein expression and quantitative binding assessment. Following binding assessment, some of the resulting mutations were combined using site directed mutagenesis into single scFv SMIP molecules. An example is the ATH105 SMIP which differs from the parental ATH064 SMIP by a single mutation in each CDR3 (VH-CDR3: S105T; and VL-CDR3: V234P).

Because the Dyax phage display system is designed for screening Fab molecules, it was unclear whether the scFv fusion to Genelll phage would retain binding. Various control molecules were generated to test the system and binding was confirmed by ELISA, demonstrating that the system did work.

To determine the effectiveness of mutant binding to human hyper IL6, ELISA plates were coated with goat anti-human IgG Fc antibody at 2ug/ml_ in PBS overnight at 4°C and blocked with 3% BSA PBS. Dilutions of human GP130 or SMIP were applied in 3x dilutions starting at 300ng/ml_ at room temperature (R/T) for 1 hour. Human hyper IL6 was applied [10ng/ml_] at R/T for 1 hour. Anti-human IL6 biotin antibody (R&D Systems Cat#BAF206) was applied [200ng/ml_] at R/T for 1 hour. Streptavidin-HRP [1 :5000 dilution] was applied at R/T for 1 hour. QuantaBlu Fluorogenic Peroxidase Substrate

(Thermo Scientific) was used for detection with an excitation/emission maxima of 325/420nm. The results of the binding as shown in Figure 4 are expressed in MFI at 420nm.

From the affinity maturation process, several affinity matured mutants were identified. In particular, the A2M1 anti-hyper IL-6 binding domain was identified. As shown in Figure 4, the A2M1 binding molecule (also referred to as ATH105) binds tightly to hyper IL-6. In some embodiments, a polypeptide or binding domain of the invention binds hyper IL6 with a binding affinity of about kD 350-500 pM, about kD 375-500 pM, about kD 400-500 pM, about kD 425- 500 pM, about kD 450-500 pM, about kD 350-475 pM, about kD 350-450 pM, about kD 350-425 pM, about kD 350-400 pM, about kD 350-375 pM, about kD 420-440 pM, about kD 425-435 pM, about kD 428-434 pM. In some

embodiments, a polypeptide or binding domain of the invention binds hyper IL6 with a binding affinity of about kD 431 pM. The sequence of the A2M1 differs from the A2 binding domain by two CDR3 substitutions: VH: S7T and VL: V10P. The amino acid sequence of the A2M1 binding domain SMIP is provided in SEQ ID NO:12. The polynucleotide sequence encoding the A2M1 SMIP binding domain is provided in SEQ ID NO:1 1 . The amino acid sequence of the A2M1 VH and VL are set forth in SEQ ID NOs: 13 and 14. The A2M1 VHCDR3 and VLCDR3 sequences which differ from the A2 parent sequence are set forth in SEQ ID NOs:15 and 16

respectively. Several other mutants with improved binding affinity (see Figure 4A) were also identified (ATH072; ATH104; ATH106; the amino acid sequences for these mutant SMIPs are provided in SEQ ID NOs: 18, 23 and 28,

respectively; the polynucleotide sequence are set forth in SEQ ID NOs:17, 22 and 27, respectively; the amino acid sequence for the VH and VL of each of these affinity matured mutants is provided in SEQ ID NOs:19, 20; 24, 25; and 29, 30, respectively). These mutants differ from the parent A2 binding domain by the following CDR3 substitutions: ATH072: vL: V10P; ATH104: vL: S7E, V10P; ATH106: vH:S7T, vL:S7E, V10P.

Blocking of IL6 or hyper IL6 induced cell proliferation of TF-I cells was examined for the affinity matured mutants. As shown in Figure 5, multiple mutants identified, in particular the A2M1 mutant, have enhanced blocking of functional activity.

In an additional experiment, the ability of A2M1 SMIP to block STAT3 phosphorylation induced by human hyper-IL6 was evaluated.

TF-1 cells (human erythroleukemia cells) were seeded at 3 x10⁵ in GM- CSF free growth medium (10% FBS-RPMI 1640; 2mM L-glutamine; 100 units/ml penicillin; 100 g/ml streptomycin; 10 mM HEPES; 1 mM sodium pyruvate) one day before use in STAT3 phosphorylation assays. For blocking HIL-6 or IL6/IL6R complex activity, serial dilutions of the SMIP/antibody/protein of interest was pre-incubated with a fixed concentration human hyper IL-6 (HIL- 6) or CHO/HEK293 derived supernatant containing human IL-6/IL-6R complex (see Example 3 below) in 96-well plates. The human soluble gp130-Fc chimeric fusion protein (R&D Systems) was used as a positive control. During this pre- incubation period (1 hour; 37°C), cells were harvested by centrifugation, washed twice, and then seeded to 96-well plates (106 cells/well) in GM-CSF free medium. To initiate the STAT assay, cells were pelleted by centrifugation, and then suspended in the cytokine mixtures described above. The final assay mixture, in a total volume of 100 μΙ/well, containing SMIP, IL6/IL6R complex or HIL-6 and cells was incubated at 37°C, 5%CO2 for 30 minutes. Following incubation, the cell were collected by centrifugation, washed with PBS, and then fixed in 100 μΙ/well of 4% paraformaldehyde for 15 minutes at room temperature. Following an additional PBS wash, the cells were permeabilized with 10ΟμΙ/well of ice-cold 100% methanol for 10 minutes at -20°C, then washed with ice-cold PBS. Cell were then stained with 5 μΙ/well of PE- conjugated phospho-STAT3 antibody (BD Biosciences) in FACS buffer (PBS containing 3% FBS) for 30 minutes on ice and analyzed by flow cytometry. The data are presented as the normalized mean fluorescence expressed as percentage of maximum sample fluorescence (from triplicate sample sets). As shown in Figure 24, the A2M1 SMIP was able to neutralize STATS

phosphorylation induced by human hyper-!L8.

In summary, A2 SMIPs block human hyper IL-6 induced proliferation of TF-1 cells without interfering with the IL-6 dependent response mediated via the membrane IL-6R. The A2M1 SMIP is more effective than A2 at blocking human hyper IL-6 induced response.

EXAMPLE 3

A2M1 SMIP BINDS TO HUMAN IL-6/SIL-6R COMPLEX

Human hyper IL-6 is a designed cytokine consisting of IL-6 and soluble IL-6R fused by a flexible peptide linker, differing from the physiological form of IL-6/slL-6R complex. This example describes experiments conducted to determine whether the A2M1 SMIP can bind to the human IL-6/slL-6R complex and whether the A2M1 blocks the bioactivity of the human complex.

An advanced in vitro bioassay system was developed to evaluate the ability of A2 SMIPs to block bioactivity of human IL-6/slL-6R complex. In an effort to obtain a more native form of the IL6-IL6R complex, a bi-cistronic vector was designed for the separate expression of hlL-6 and hlL-6R in mammalian cells. The vector was transiently transfected into HEK293 cells. In this system, both hlL-6 and hlL-6R are secreted into the media. As shown in Figure 6, the slL-6 and IL-6R in the supernatant (ZAR067) were able to form a complex that is bound by human gp130.

Further experiments showed that the parent A2 SMIP can bind to the native complex (see Figure 7). As discussed above, the A2M1 was selected for its increased binding to hyper IL6; it's binding to hyper IL6 was found to be 4-5 times better than the A2 SMIP (see Figure 8). However, when comparing the binding of the in vitro generated "native" complex, the A2M1 binding was shown to be at least 7.5-fold greater than the parent A2 SMIP (see Figure 9A). EC50s were calculated by the program GraphPad Prism 5 with A2M1 and A2 being 0.08 pm/ml and 0.6 pm/ml, respectively, which is a 7.5-fold difference. These experiments were confirmed using a similar a CHO cell line. In particular, a CHO lines was engineered to secrete, individually, human IL-6 and human IL- 6R. A 10-day culture supernatant was used in an ELISA to assess binding of the IL6/IL6R complex found in the supernatant by the A2 and A2M1 SMIPs. Briefly, in a 96-well ELISA plate, the A2 and A2M1 SMIPs were serially diluted 1 :3, starting at 7.5 pmoles/ml. 10Oul of supernatant was added to each well and incubated for 1 hour at room temperature. A biotinylated anti-human IL6 antibody was used for the detection of IL6+IL6R complex binding. Results are shown in Figure 9B. EC50s were calculated by the program GraphPad Prism 5 with A2M1 and A2 being 72 fmoles/ml and 630 fmoles/nnl, respectively, which is a 8.8-fold difference.

In addition, A2M1 was shown to bind better than two other matured variants of the A2 SMIP (see Figure 10; ATH104 and ATH106). EC50s were calculated by the program GraphPad Prism 5 with A2M1 and A2 being 0.10 pm/ml and 1 .75 pm/ml, respectively, which is a 17.5-fold difference. These two mutants have the following substitutions in the CDR3 regions of the VH and VL: ATH104: VL: S7E, V10P; ATH106: VH: S7T; VL: S7E, V10P (see alignment provided in Figure 23 and SEQ ID NOs: 24, 25, 29 and 30).

Another in vitro bioassay system was developed to evaluate the ability of the A2 SMIPs to block the bioactivity of human IL-6/slL-6R complex. In particular, the BAF3 mouse pro B cell line was used. This is an IL-3 dependent cell line that does not express either mlL-6R or mgp130. Three transfectant cell lines were established from the BAF3 cells: the first cell line was transfected with hlL-6R, the second with hgp130 and the third with both hlL-6R and hgp130. Expression was confirmed by flow cytometry. BAF3 cell lines also proliferate in response to hyper IL-6 (see Figure 1 1 ) and in response to recombinant non-mammalian derived IL-6/slL-6R (see Figure 12). These data show that BAF3 cells transfected with hgp130 will only proliferate if hlL-6 and hlL-6R are present, either added as a complex (HIL-6) or when the individual components are present to form a complex (Figure 1 1 B, 12A, 12C). The BAF3/hgp130 cell lines will not proliferate in response to either hlL-6 (Figure 1 1 A, 12A) or hlL-6R alone (Figure 12B). In addition, Figure 13 shows the proliferation of BAF3 cell lines transfected with hgp130 in response to

mammalian-derived human IL-6/slL-6R complex (ZAR067 supernatant).

ZAR067 supernatant is from HEK293 cells transfected with hlL-6 and hlL-6R which are secreted separately to form IL-6/slL-6R complex. Therefore, this BAF3 cell line transfected with hgp130 is a unique in vitro system for measuring IL-6 trans-signaling activity.

Once the in vitro bioassay system was established, the bioactivity of the A2 SMIPs was evaluated. As shown in Figure 14, the A2 and A2M1 SMIPs neutralized the human IL-6/slL-6R induced proliferation of BAF3/hgp130 cells. The A2M1 SMIP was effective with both mammalian-derived (ZAR067 supernatant) and non-mammalian derived complexes while the A2 SMIP was less potent.

The ability of A2M1 SMIP to block STAT3 phosphorylation induced by HEK-293-produced human IL-6/IL-6R complex or by CHO-produced human IL- 6/IL-6R complex was then evaluated in a STAT3 phosphorylation assay as described in Example 2. The graph shown in Figure 25 represents the normalized data of FI measured in triplicate and demonstrates that the A2M1 SMIP neutralizes STAT3 phosphorylation induced by the human IL-6/IL-6R complex. The graph shown in Figure 26 represents the normalized data of MFI measured in triplicate and demonstrates that A2M1 blocks STAT3

phosphorylation induced by CHO-produced human IL-6/IL-6R complex.

A native, human-derived IL-6/slL-6R complex was then generated from human plasma from whole blood treated with LPS. As shown in Figure 15, BAF3/hgp130 cells proliferated in response to the native human-derived IL- 6/slL-6R complex from LPS treated plasma. Additionally, as shown in Figure 16, native human-derived IL-6/slL-6R complex-induced proliferation of

BAF3/hgp130 cells was neutralized by rhgp130-Fc and an anti-hlL-6 antibody (MQ2-13A5). Next, the ability of the A2 and A2M1 SMIPs to neutralize native human-derived IL-6/slL-6R complex-induced proliferation of BAF3/hgp130 cells was tested. As shown in Figure 17, A2M1 SMIP effectively neutralized native human-derived IL-6/slL-6R complex-induced proliferation of BAF3/hgp130 cells, and the A2 SMIP was considerably less potent than the A2M1 SMIP.

EXAMPLE 4

BINDING SITE CHARACTERIZATION OF A2M1 SMIP This Example describes experiments conducted to characterize the binding site of the A2M1 binding domain.

Like A2, A2M1 cannot bind IL-6 and can bind IL-6R at high

concentrations (see Figure 18). In order to further characterize the A2 and A2M1 binding site, binding studies were carried out using a panel of previously characterized antibodies with known binding sites. In particular, as summarized in the diagram in Figure 19, IL-6 assembles with IL-6R and gp130 to form a trimeric complex. Site I and II drive complex formation. The IL-6 signaling complex is a hexamer. Site III drives dimerization and subsequent signaling. As demonstrated in Figure 20, antibodies known to bind to site III {e.g., AH65^* and CLB-16) effectively competed with the A2 binding domain for binding to the IL-6/slL-6R. The C3 binding domain was previously shown to have a non- overlapping binding site with A2. Thus, this experiment showed that A2 binds Site III of the IL-6/IL-6R complex, blocking biological signal but not the assembly of sgpl 30 into the trimeric complex.

In an additional experiment, a variety of H-IL6 mutants were constructed as summarized in Figure 21 and Table 3. ZAR041 is the parental H-IL6 on which the mutations were made. The sequence of the H-IL6 used for these experiments is provided in SEQ ID NO:750. The leader sequence is amino acids 1 -22 of SEQ ID NO:750. The positions mutated refer to the mature protein without the leader peptide. The mature protein without the leader peptide is set forth in SEQ ID NO:749. These mutations did not affect the biological function of the H-IL6 molecules. Thus, these mutant molecules retain the ability to induce proliferation and STAT3 phosphorylation in in vitro assays. Comparison binding studies between A2 and A2M1 showed that A2M1 binds an epitope that is similar to A2 but that is distinct. In particular, as shown in Figure 22A and 22B, positions F134, 1170 and R132 are important for A2 binding as mutations at these positions resulted in reduced A2 binding.

However, the A2M1 binding domain was still able to bind molecules having mutations at these positions (see Figure 22C). Mutations at these positions also did not affect binding of the previously characterized antibody AH-65.

Table 3: mutants of hyper IL-6 to characterize A2 and A2M1 binding epitopes.

ZAR058 X X

ZAR059 X X X X X X

ZAR062 X

ZAR063 X X

ZAR064 X

ZAR065 X X

EXAMPLE 5

TESTING A2M 1 SM IP IN BLOCKING BIOACTIVITY OF NATIVE HUMAN-DERIVED IL-6/SIL-

6R COMPLEX To test the ability of an A2M1 SMIP in blocking bioactivity of native human-derived IL-6/slL-6R complex, BAF3/hgp130 cells were incubated in 96 well plates for 72 h in the presence of human LPS plasma (1 :8 dilution) and various concentrations of A2M1 SMIP or rhgp130-Fc. Proliferation of

BAF3/hgp130 cells was measured by a ³H-thymidine incorporation assay. The results are expressed as mean of cpm ± SD of duplicates and IC50 values.

Table 4 and Figure 27 shows an A2M1 SMIP (closed circle) effectively blocked a native human-derived IL-6/IL-6R complex induced proliferation of BAF3/hgp130 cells.

Table 4

EXAMPLE 6

Bi-sPECiFic BINDING MOLECULE WITH BOTH A TNFR DOMAIN AND AN IL-6/SIL-6R

COMPLEX BINDING DOMAIN

A bispecific molecule was designed to bind both IL-6/SIL-6R complex and TNF. This bispecific molecule comprised from N-terminus to C-terminus a fragment of a TNFR2 that binds TNF-a (amino acids 23-257 of SEQ ID NO:1 18), a lgG1 Fc domain comprising amino acids 250-480 of SEQ ID NO:12, a linker as set forth in SEQ ID NO:330 and an IL-6/IL-6R complex binding domain (amino acids 1 -247 of SEQ ID NO:12. The complete amino acid sequence of this bispecific molecule and a corresponding nucleotide coding region are set forth in SEQ ID NOs:752 and 751 , respectively.

This molecule was expressed using the signal sequence of SEQ ID NO:735.

EXAMPLE 7

Bi-sPECiFic BINDING MOLECULE WITH BOTH A TNF-a BiNDiNg DOMAIN AND AN

IL-6/SIL-6R COMPLEX BINDING DOMAIN

A bispecific molecule was designed to bind both IL-6/SIL-6R complex and TNF. This bispecific molecule comprised from N-terminus to C-terminus a scFv that binds TNF-a (comprising amino acids 23-265 of SEQ ID NO:754), a lgG1 Fc domain amino acids 250-480 of SEQ ID NO:12, a linker as set forth in SEQ ID NO:330 and an IL-6/SIL-6R complex binding domain comprising amino acids 1 -247 of SEQ ID NO:12. The complete amino acid sequence of this bispecific molecule and a corresponding nucleotide coding region are set forth in SEQ ID NOs:754 and 753, respectively.

This molecule was expressed using the signal sequence of SEQ ID NO:735.

In summary, the above Examples demonstrate that the A2M1 binding domain binds hyper IL-6 and native human forms of IL-6/slL-6R complex and blocks biological activity of the native complex without significantly interfering with IL-6 cis-signaling. Therefore, fusion proteins comprising the A2M1 binding domain are useful in a variety of therapeutic settings for the treatment of disorders associated with aberrant IL-6 expression, activity and/or signaling.

Claims

CLAIMS What is claimed is:

1 . An isolated polypeptide comprising a binding domain that binds a soluble IL6/IL6R (slL6xR) complex, wherein the isolated polypeptide:

(a) binds to the slL6xR complex with a higher affinity than either IL6 alone or IL6Ra alone;

(b) competes with membrane gp130 for binding to the slL6xR complex;

(c) binds to human native slL6xR about 5-25 fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2; and

(d) the polypeptide is not a gp130.

2. The isolated polypeptide of claim 1 , wherein the isolated polypeptide binds to IL6Ra alone with a higher affinity than to IL6 alone.

3. The isolated polypeptide of any one of the previous claims, wherein the isolated polypeptide preferentially inhibits IL6 trans-signaling over IL6 cis- signaling.

4. The isolated polypeptide of any one of the previous claims, wherein the isolated polypeptide inhibits the biological activity of a human native slL6xR complex.

5. The isolated polypeptide of claim 4, wherein the inhibition of the biological activity of a human native slL6xR complex is statistically significantly greater as compared to an isolated polypeptide with an amino acid sequence consisting of SEQ ID NO:2.

6. The isolated polypeptide of claim 4, wherein the biological activity is measured by cell proliferation or STAT3 phosphorylation induced by the human native slL6xR complex.

7. The isolated polypeptide of claim 6, wherein the cell proliferation comprises proliferation of a BAF3 cell line expressing gp130.

8. The isolated polypeptide of claim 1 , wherein the isolated polypeptide binds to human native slL6xR about 5-25 fold better in an ELISA than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2.

9. The isolated polypeptide of claim 1 , wherein the binding domain that binds slL6xR comprises:

(a) a VL region comprising a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ;

(b) a VH region comprising a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or

(c) a VL of (a) and a VH of (b).

10. An isolated polypeptide comprising a binding domain that binds slL6xR wherein the binding domain comprises:

(a) a VL region comprising a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ;

(b) a VH region comprising a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or

(c) a VL of (a) and a VH of (b).

1 1 . The isolated polypeptide of claim 10, wherein VL CDR3 is SEQ ID NO:16 and the VH CDR3 is SEQ ID NO:15.

12. An isolated polypeptide that binds to a slL6xR wherein the isolated polypeptide comprises from amino-terminus to carboxy-terminus:

(a) a binding domain, wherein the binding domain binds to human native slL6xR about 5-25 fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2; (b) an immunoglobulin heavy chain CH2 constant region polypeptide, and

(c) an immunoglobulin heavy chain CH3 constant region polypeptide, wherein the isolated polypeptide inhibits the biological activity of the human native slL6xR complex.

13. The isolated polypeptide of claim 12, comprising a hinge region between (a) and (b).

14. The isolated polypeptide of claim 12 or 13 wherein the binding domain comprises:

(a) a VL region comprising a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ;

(b) a VH region comprising a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or

(c) a VL of (a) and a VH of (b).

15. The isolated polypeptide of claim 14, wherein VL CDR3 is SEQ ID NO:16 and the VH CDR3 is SEQ ID NO:15.

16. The isolated polypeptide of claim 10 or 14, wherein the VL region comprises the amino acid sequence of SEQ ID NO:14.

17. The isolated polypeptide of claim 10 or 14, wherein the VH region comprises the amino acid sequence of SEQ ID NO:13.

18. The isolated polypeptide of claim 10 or 14, wherein the VL region comprises the amino acid sequence of SEQ ID NO:14 and the VH region comprises the amino acid sequence of SEQ ID NO:13.

19. The isolated polypeptide of claim 10 or 14, wherein the VL and VH regions are humanized.

20. The isolated polypeptide of any one of claims 1 -1 1 wherein the isolated polypeptide is an antibody or an antigen-binding fragment thereof or a polypeptide comprising an antigen-binding fragment thereof.

21 . The isolated polypeptide of claim 20, wherein the antibody or antigen-binding fragment thereof is non-human, chimeric, humanized or human.

22. The isolated polypeptide of claim 20, wherein the antibody or antigen-binding fragment thereof comprises a VL region selected from the group consisting of an amino acid sequence as set forth in SEQ ID NOS:14, 20, 25 and 30.

23. The isolated polypeptide of claim 20, wherein the antibody or antigen-binding fragment thereof comprises a VH region selected from the group consisting of an amino acid sequence as set forth in SEQ ID NOS:13, 19, 24 and 29.

24. The isolated polypeptide of claim 20 wherein the antibody or antigen-binding fragment thereof comprises a VL region comprising the amino acid sequence as set forth in SEQ ID NO:14 and a VH region comprising the amino acid sequence as set forth in SEQ ID NO:13.

25. The isolated polypeptide of any one of claims 1 -1 1 , wherein the isolated polypeptide or binding domain is selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an scFv, a dAb, and an Fv fragment.

26. The isolated polypeptide of claim 25, wherein the scFv comprises the amino acid sequence provided in amino acids 1 -247 of SEQ ID NO: 12.

27. The isolated polypeptide of claim 12, wherein the binding domain comprises an scFv comprising amino acids 1 -247 of SEQ ID NO: 12.

28. The isolated polypeptide of claim 12, wherein the isolated

polypeptide or binding domain is non-human, chimeric, humanized or human.

29. The isolated polypeptide of claim 12, comprising a hinge region having an amino acid sequence of any one of SEQ ID NOS:37-70.

30. The isolated polypeptide of claim 12, wherein the CH2 and the CH3 domains comprise an immunoglobulin CH2 and a CH3 domain of lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2 or IgD.

31 . The isolated polypeptide of claim 30, wherein the immunoglobulin CH2 and CH3 domains comprise human lgG1 CH2 and CH3 domains.

32. The isolated polypeptide of claim 31 , wherein the human lgG1 CH2 domain comprises the amino acid sequence of SEQ ID NO:96 and the human lgG1 CH3 domain comprises the amino acid sequence of SEQ ID NO:436.

33. The isolated polypeptide of claim 12, wherein the isolated

polypeptide comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOS:12, 18, 23 and 28.

34. An isolated polypeptide comprising a binding domain which binds to a mutated Site III epitope of the IL-6 signaling complex, wherein the mutated Site III epitope comprises at least mutations at one or two positions selected from F134, 1170 and R132 of the IL6R portion of the hyperlL6 fusion protein as set forth in SEQ ID NO:749.

35. The isolated polypeptide of any one of claims 1 -19 and 26-34, wherein the isolated polypeptide is contained in a single-chain multi-specific binding protein comprising an Fc region constant domain disposed between a first binding domain and a second binding domain, wherein the first binding domain is a slL6xR binding domain according to claims 1 -19 and 26-34 and the second binding domain is a slL6xR binding domain or is a binding domain that specifically binds a target molecule other than a slL6xR.

36. The isolated polypeptide of claim 35 wherein the second binding domain is a tumor necrosis factor (TNF) antagonist or a transforming growth factor beta (TGF ) binding domain.

37. The isolated polypeptide of claim 36, wherein the TNF antagonist is a TNFR ectodomain or a binding domain that specifically binds TNFa.

38. The isolated polypeptide of claim 37, wherein the binding domain that specifically binds TNFa comprises amino acids 23-141 of SEQ ID NO:754, amino acids 159-265 of SEQ ID NO:754, amino acids 23-141 and 159-265 of SEQ ID NO:754 or amino acids 23-265 of SEQ ID NO:754.

39. The isolated polypeptide of claim 37, wherein the TNFR ectodomain is a TNFR1 or TNFR2 ectodomain.

40. The isolated polypeptide of claim 37, wherein the TNFR ectodomain comprises amino acids 23-163 of SEQ ID NO:1 18, amino acids 23-185 of SEQ ID NO:1 18, amino acids 23-235 of SEQ ID NO:1 18, amino acids 23-257 of SEQ ID NO:1 18 or amino acids 31 -21 1 of SEQ ID NO:1 19.

41 . The isolated polypeptide of claim 37, wherein the TNFR ectodomain comprises:

(i) amino acids 23-163 of SEQ ID NO:1 18 with a deletion of amino acid glutamine at position 109;

(ii) amino acids 23-185 of SEQ ID NO:1 18 with a deletion of amino acid glutamine at position 109 and a deletion of amino acid proline at position 131 ; or

(iii) amino acids 23-235 of SEQ ID NO:1 18 with a deletion of amino acid glutamine at position 109, a deletion of amino acid proline at position 131 , and a substitution of amino acid aspartate at position 257.

42. The isolated polypeptide of claim 35, wherein the Fc region constant domain comprises lgG1 CH2 and CH3 domains.

43. The isolated polypeptide of claim 35, wherein the Fc region constant domain is disposed between a first linker peptide and a second linker peptide.

44. The isolated polypeptide of claim 43, wherein the first and second linker peptides are independently selected from the linkers provided in SEQ ID NOS:193-360.

45. The isolated polypeptide of claim 43 or 44 wherein the first or second linker peptide comprises an interdomain region of an immunoglobulin super family member.

46. The isolated polypeptide of claim 43 or 44 wherein the first or second linker peptide comprises an immunoglobulin hinge region.

47. An isolated polypeptide comprising the following structure:

N-BD1 -L1 -CH2CH3-L2-BD2-C wherein:

N is the amino-terminus,

C is the carboxy terminus,

BD1 comprises a binding domain that binds slL6xR or a target molecule other than slL6xR;

L1 is a first linker peptide;

-CH2CH3- comprises an immunoglobulin CH2 and CH3 constant region; L2 is a second linker peptide;

BD2 is a binding domain that specifically binds slL6xR or a target molecule other than slL6xR; and

at least one of BD1 or BD2 is a binding domain that binds to human native slL6xR about 5-25 fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2.

48. The isolated polypeptide of claim 47, wherein the L1 is an

immunoglobulin lgG1 hinge region having an amino acid sequence selected from the group consisting of SEQ ID NOs:120-192.

49. The isolated polypeptide of claim 47 or 48, wherein the -CH2CH3- comprises human lgG1 CH2 and CH3 domains.

50. The isolated polypeptide of claim 47 or 48, wherein the -CH2CH3- comprises variant human lgG1 CH2 and CH3 domains lacking one or more effector functions.

51 . The isolated polypeptide of anyone of claims 47-50, wherein the L2 is a linker peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS:193-360 and 745-748.

52. The isolated polypeptide of anyone of claims 47-51 , wherein the BD1 is a TNF antagonist or a TGF antagonist and the BD2 is a binding domain that specifically binds the human slL6xR complex.

53. The isolated polypeptide of anyone of claims 47-51 , wherein the BD2 is a TNF antagonist or a TGF antagonist and the BD1 is a binding domain that specifically binds the human slL6xR complex.

54. The isolated polypeptide of anyone of claims 47-53, wherein the isolated polypeptide binds to human native slL6xR about 5-25 fold better in an ELISA than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2.

55. The isolated polypeptide of anyone of claims 47-54, wherein BD2 comprises:

(a) a VL region comprising a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ;

(b) a VH region comprising a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or

(c) a VL of (a) and a VH of (b).

56. An isolated polypeptide comprising:

an Fc region constant domain;

a hinge region disposed C-terminal to the Fc region constant domain; and

a binding domain, wherein the binding domain binds to human native slL6xR about 5-25 fold better than a polypeptide with an amino acid sequence consisting of SEQ ID NO:2.

57. The isolated polypeptide of claim 56, wherein the isolated

polypeptide inhibits the biological activity of the human native slL6xR complex.

58. The isolated polypeptide of claim 56 or 57, wherein the binding domain comprises:

(a) a VL region comprising a CDR1 amino acid sequence of SEQ ID NO:8, a CDR2 amino acid sequence of SEQ ID NO:9, and a CDR3 amino acid sequence selected from SEQ ID NOs:16, 21 , 26 and 31 ;

(b) a VH region comprising a CDR1 amino acid sequence of SEQ ID NO:5, a CDR2 amino acid sequence of SEQ ID NO:6, and a CDR3 amino acid sequence selected from SEQ ID NOs:15 and 7; or

(c) a VL of (a) and a VH of (b).

59. The isolated polypeptide of any one of the previous claims, wherein the polypeptide is contained in a first single chain polypeptide comprising a first heterodimerization domain that is capable of associating with a second single chain polypeptide comprising a second heterodimerization domain, wherein the first and second heterodimerization domain is different and wherein the associated first and second single chain polypeptides form a polypeptide heterodimer.

60. A composition comprising the isolated polypeptide according to any one of claims 1 -59 and a pharmaceutically acceptable excipient.

61 . An expression vector capable of expressing the isolated polypeptide of any one of claims 1 -59.

62. An isolated host cell comprising the expression vector of claim 61 .

63. A method for treating an inflammatory disorder comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 60.

64. The method of claim 63, wherein the inflammatory disorder is selected from the group consisting of rheumatoid arthritis, psoriasis, colitis, ulcerative colitis, Crohn's disease, and cardiovascular disease.

65. A method for treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 60.

66. The method of claim 65, wherein the cancer is selected from the group consisting of colon cancer; ovarian cancer; pancreatic cancer; prostate cancer, non-Hodgkin's lymphoma, kidney cancer, lung cancer.