Interleukin 11 polypeptides and methods of use

Abstract

Provided are IL-11 polypeptides. In certain embodiments, an IL-11 polypeptide of the present disclosure comprises one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 receptor (IL-11R) as compared to wild-type human IL-11. According to some embodiments, such an IL-11 polypeptide further comprises one or more amino acid substitutions which inhibit formation of a complex comprising the IL-11 polypeptide, human IL-11R and glycoprotein 130 (gp130) upon binding of the IL-11 polypeptide to human IL-11R. Also provided are methods of using the IL-11 polypeptides of the present disclosure. For example, provided are methods of treating a condition associated with IL-11R signaling in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising an IL-11 polypeptide of the present disclosure. Non-limiting examples of such conditions include cancer, fibrosis, etc. associated with IL-11R signaling.

Description

[0001] INTERLEUKIN 1 1 POLYPEPTIDES AND METHODS OF USE

[0002] CROSS-REFERENCED TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 521 ,277, filed June 15, 2023, which application is incorporated herein by reference in its entirety.

[0004] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0005] PROVIDED AS A SEQUENCE LISTING XML FILE

[0006] A Sequence Listing is provided herewith as a Sequence Listing XML, “STAN-2094WO_SEQLIST”, created on June 14, 2024 and having a size of 4,160 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

[0007] INTRODUCTION

[0008] Cytokines play an essential role in cell-to-cell signaling processes across nearly all systems in the body.1The interleukin-6 (IL-6) family of cytokines includes IL-6, IL-1 1 , IL-27, cardiotrophin-like cytokine factor 1 (CLCF1 ), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and oncostatin M (OSM).2These cytokines are comprised of four-helical bundles and share the signaling co-receptor glycoprotein 130 (gp130) which is ubiquitously expressed throughout the body.3Many of the IL-6 family ligands and co-receptors have been targets of pre-clinical and clinical therapeutic campaigns for cancer and autoimmunity.4 8

[0009] The pleiotropic cytokine, IL-1 1 , has been implicated in a variety of diseases across tissue types.9IL-1 1 is a non-glycosylated 178-amino-acid soluble cytokine with a molecular weight of ~19 kDa. IL-1 1 has been proposed to first bind to the IL-11 receptor (IL-11 R), and along with gp130 forms a hexameric complex via two independent IL-1 1 -gp130 binding sites.10This complex promotes downstream Janus kinase (JAK) activation and signaling through signal transducer and activator of transcription 3 (STAT3), phosphoinositide 3-kinase (PI3K), and extracellular signal- regulated kinase (ERK).r 13Increased IL-1 1 signaling has been associated with cancer proliferation, metastasis, and chemoresistance,14 18leading to the progression of gastric, colorectal, breast, and bone cancers among others.19 22IL-11 has also been implicated in fibrotic remodeling that affects cancer associated fibroblasts as well as lung, liver, and heart fibrosis.18,2326As a result of these disease associations, the IL-1 1 axis is currently being targeted by clinical stage antibodies for pulmonary fibrosis.2728Additionally, IL-1 1 has been identified as a therapeutic target for lung adenocarcinoma.29’30 SUMMARY

[0010] Provided are IL-11 polypeptides. In certain embodiments, an IL-11 polypeptide of the present disclosure comprises one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-1 1 receptor (IL-11 R) as compared to wild-type human IL-11 . According to some embodiments, such an IL-1 1 polypeptide further comprises one or more amino acid substitutions which inhibit formation of a complex comprising the IL-11 polypeptide, human IL-11 R and glycoprotein 130 (gp130) upon binding of the IL-11 polypeptide to human IL- U R. Also provided are methods of using the IL-1 1 polypeptides of the present disclosure. For example, provided are methods of treating a condition associated with IL-1 1 R signaling in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising an IL-1 1 polypeptide of the present disclosure. Non-limiting examples of such conditions include cancer, fibrosis, etc. associated with IL-1 1 R signaling.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 : Sorting yeast-displayed IL-11 libraries against human IL-11 receptor, (a) Schematic of yeast surface-displayed IL-1 1 binding to soluble his-tagged IL-1 1 R detected by fluorescent antibodies (blue: c-myc tag for IL-1 1 expression; red: IL-1 1 R binding detected by 6x histidine tag), (b) Three rounds of fluorescence activated cell sorting against indicated concentrations of IL-1 1 R. Polygons on cell dot plots indicate gates set to collect the yeast population that was used in the subsequent sort, (c) Improvement in binding of yeast from the final sort rounds to 1 nM IL-11 R compared to initial library 1 and 2. (d) Table of amino acid mutations from sequencing eight clones from post 2.3 and 2.4 sorts. The wild-type IL-1 1 amino acid residue is indicated along the top. Consensus mutations are highlighted in gray.

[0013] FIG. 2: Characterizing IL-11 variants with increased affinity for human IL-11 receptor, (a) Table of individual variants along with their measured apparent affinities for IL-1 1 R compared to wild-type (WT) IL-1 1 ; variants are subsequently named after their mutations, (b) Dose-dependent binding curve of human IL-11 R to yeast-displayed IL-1 1 GIPR and IL-11 WT measured by flow cytometry. Points represent the fraction bound to IL-11 R of the expressing population + / - SD, n = 3, curve fit by non-linear regression, (c) IL-1 1 crystal structure (PDB: 6040) with proposed IL-1 1 R binding residues (Site I) and GIPR mutations, (d) Phospho-STAT3 signaling measured by luminescence in HeLa reporter cells transduced with IL-11 R. IL-11 GIPR EC50 = 24 + / - 4 pM; IL-11 WT EC50 = 140 + / - 30 pM. Points represent mean + / - SD, n = 3, curve fit by non-linear regression, (e) Western blot of A549 lysates for phospho-STAT3 (Y705) with indicated concentration of IL-1 1 WT or IL-11 GIPR. Tubulin and STAT3 as loading controls. Right panel: Quantified pSTAT3 / Tubulin levels, mean + / - SD, n = 2. *P < 0.05 by unpaired t-test.

[0014] FIG. 3: Engineering IL-11 GIPR for loss of binding to gp130. (a) Schematic of trimeric complex of yeast surface-displayed IL-1 1 GIPR binding to soluble IL-1 1 R and gp130 detected by fluorescent antibodies (blue: c-myc tag for IL-1 1 GIPR expression; red: gp130 binding), (b) Yeast- displayed IL-1 1 GIPR library dot plots showing alternating two negative flow cytometry sorts against gp130 and one positive sort with IL-1 1 R at the indicated concentrations. Polygons indicate gates set to collect the yeast population that was used in the subsequent sort, (c) Table of amino acid mutations identified from sequencing eight clones after Sort 6. The wild-type IL-1 1 amino acid residue is indicated along the top. L24R and W147A are present in all eight clones, (d) Normalized binding of yeast-displayed IL-1 1 mutants to varying concentrations of gp130-Fc with 100 nM IL-1 1 R added to all samples. Data points represent geometric mean of fluorescence of the expressing population + / - SD, n = 3. (e) Schematic of IL-11 / IL-11 R / gp130 hexameric complex with IL-1 1 Site I, III, and III labelled, (f) Crystal structure of IL-11 (PDB: 6040) with purported Site I (IL-1 1 R binding residues) in purple and purported Site II and Site III (gp130 binding residues) in green and blue respectively. L24R and W147A are shown in red.

[0015] FIG. 4: Affinity maturation of IL-11 GIPR for mouse IL-11 R. (a) Binding of mlL-11 R to yeast-displayed mlL-11 WT (apparent Kd= 3.1 + / - 0.8 nM); IL-1 1 WT (apparent Kd= 2.2 + / - 0.3 nM); or IL-11 GIPR (apparent Kd= 1 .5 + / - 0.1 nM). Data points represent the fraction bound to IL-11 R of the expressing population + / - SD, n = 3, curve fit with non-linear regression, (b) Binding of yeast-displayed IL-11 GIPR and GIPR L24R W147A pre-complexed with 100 nM mlL-11 R to mouse gp130-Fc. Data points are geometric mean of fluorescence of the expressing population + / - SD, n = 3. (c) Binding curve of yeast-displayed IL-11 GIPR S60T to mlL-1 1 R by yeast surface display. Data points represent the fraction bound to gp130-Fc of the expressing population + / - SD, n = 3, curve fit with non-linear regression, (d) Crystal structure of IL-11 (PDB: 6040) with the mutations that comprise enlL-11 highlighted based on function in the indicated colors.

[0016] FIG. 5: enlL-11 inhibits IL-11 mediated cell signaling and slows tumor growth in vivo, (a) Phospho-STAT3 signaling measured by luminescence in HeLa reporter cells transduced with IL-1 1 R and luciferase under control of a STAT3 inducible response element. 0.5 nM of wild-type (WT) IL-11 was added to all samples along with varying concentrations of WT IL- 1 1 L24R W147A mutations (IL-1 1 RA) or enlL-11 . Data points represent mean + / - SD, n = 3. (b) Western blot of A549 lysates probed for phospho-STAT3 (Y705) in response to 0 or 20 nM WT IL-11 and the indicated concentrations of IL-1 1 RA (left) or enlL-1 1 (right). Tubulin and STAT3 as loading controls. Right panel: Quantified pSTAT3 / Tubulin levels normalized to the positive control, mean + / - SD, n = 3. *P < 0.05, **P < 0.01 by unpaired t-test. (c) Nude mice bearing A549 flank tumors treated with 5 mg / kg of enlL-1 1 (n = 7) or saline (n = 7) three times a week for four weeks. Points are mean + / - SE. **P < 0.01 by two-way ANOVA. Final fold change in volume for each tumor plotted on the right. *P < 0.05 by unpaired t-test. (d) Representative hematoxylin and eosin (H&E) staining and phospho-histone H3 (PH3) immunohistochemistry for saline and enlL- 1 1 -treated tumors. Scale bars, 100 pm. Quantified positive nuclei were compared using unpaired t-test (n = 6, **P < 0.01). Error bars represent ± SD. Figure 6: Characterization of first generation IL-11 library clones, (a) Table of amino acid mutations identified from sequencing eight clones after sort 1 .3 of yeast-displayed Library 1 , generated by error-prone PCR. The wild-type (WT) IL-1 1 amino acid residue is indicated along the top. L57P, highlighted in gray, is seen in 4 out of 8 clones, (b) Binding curve of yeast-displayed IL-11 L57P compared to WT IL-11 as measured by flow cytometry. Data points represent fraction bound to IL-11 R of the expressing population + / - SD, n = 3, curve fit by non-linear regression.

[0017] Figure 7: Second generation IL-11 library sorting and characterization, (a) Dot plots representing four rounds of fluorescence activated cell sorting of yeast-displayed IL-1 1 library 2 (generated by DNA shuffling and error-prone PCR) against indicated concentrations of IL-11 R. Polygons indicate gates set to collect the yeast population that was used in the subsequent sort, (b) Dose dependent IL-11 R binding curves of yeast-displayed variants and WT IL-1 1 measured by flow cytometry. Data points represent fraction bound to IL-11 R of the expressing population + / - SD, n = 3, curve fit by non-linear regression.

[0018] Figure 8: Affinity maturation of IL-11 GIPR for mouse IL-11 R. (a) Dot plots representing five rounds of fluorescence activated cell sorting of yeast-displayed IL-1 1 library against indicated concentrations of mlL-11 R. Polygons indicate gates set to collect the yeast population that was used in the subsequent sort, (b) Table of amino acid mutations identified from sequencing eight clones from post fourth and fifth round sorts. The wild-type IL-1 1 amino acid residue is indicated along the top. Consensus mutations are highlighted in gray.

[0019] Figure 9: enlL-11 binds IL-11 R and mouse IL-11 R more tightly via biolayer interferometry (BLI). (a) Three-fold serial dilutions with the highest concentration labeled for binding of WT IL-1 1 and enlL-1 1 to IL-1 1 R. (b) Three-fold serial dilutions with the highest concentration labeled for binding of WT IL-11 and enlL-1 1 to mlL-1 1 R.

[0020] Figure 10: enlL-11 reduces phospho-ERK signaling on A549 cells, (a) Western blot of A549 lysates probed for phospho-ERK (T202 / Y204) in response to 0 or 1 0 nM WT IL-11 and the indicated concentrations of IL-1 1 RA or enlL-11 . STAT3 and ERK as loading controls. Right panel: Quantified pERK / STAT3 levels normalized to the positive control, mean + / - SD, n = 3. **P < 0.01 , ***P < 0.001 by unpaired t-test.

[0021] Figure 11 : enlL-11 reduces A549 tumor growth in dose dependent manner, (a) Initial tumor volumes of the three treatment groups, (b) Change in weights shown as a percentage of starting weight for the three treatment groups. Data points represent mean + / - SD. (c) Nude mice with A549 flank tumors treated with 5 mg / kg of enlL-1 1 (n = 7), 1 mg / kg of enlL-1 1 (n = 7), or saline (n = 7) three times a week for four weeks. Data points represent mean + / - SE. **P < 0.01 by two-way ANOVA. (d) Individual volume traces for the three treatment groups over the course of the study.

[0022] Figure 12: Comparison of IL-11 GIPR and previously engineered IL-11 mutein. (a) Sequence alignment of wild-type (WT) IL-1 1 (SEQ ID NO:1 ), IL-11 GIPR (SEQ ID NO:2), and IL- 1 1 mutein (SEQ ID NO:3), with differences highlighted, (b) Dose dependent binding curve of human IL-11 R to yeast-displayed IL-1 1 mutein, WT IL-11 , and IL-11 GIPR as measured by flow cytometry. IL-1 1 mutein apparent Kd= 1 .6 + / - 0.3 nM. Data points represent fraction bound to IL- 11 R of the expressing population + / - SD, n = 3, curve fit by non-linear regression, (c) Dose dependent binding curve of mouse IL-1 1 R to yeast-displayed IL-1 1 mutein as measured by flow cytometry. IL-1 1 mutein apparent Kd= 2.0 + / - 0.2 nM. Data points represent fraction bound to mouse IL-1 1 R of the expressing population + / - SD, n = 3, curve fit by non-linear regression.

[0023] Figure 13: Comparison of IL-11 and related IL-6 family member CLCF1. (a) Crystal structure (PDB: 6040) of IL-1 1 with purported Site I (IL-1 1 R binding residues) in purple and Site II and Site III (gp130 binding residues) in green and blue respectively. L24R and W147A are shown in red. Alpha fold predicted structure of CLCF1 with mutations found to disrupt gp130 binding shown in green and mutations found to disrupt LIFR binding shown in orange.8,54

[0024] DETAILED DESCRIPTION

[0025] Before the polypeptides, compositions and methods of the present disclosure are described in greater detail, it is to be understood that the polypeptides, compositions and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the polypeptides, compositions and methods will be limited only by the appended claims.

[0026] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the polypeptides, compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the polypeptides, compositions and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the polypeptides, compositions and methods.

[0027] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the polypeptides, compositions and methods belong. Although any polypeptides, compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the polypeptides, compositions and methods, representative illustrative polypeptides, compositions and methods are now described.

[0029] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present polypeptides, compositions and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0030] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0031] It is appreciated that certain features of the polypeptides, compositions and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the polypeptides, compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present polypeptides, compositions and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0032] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. INTERLEUKIN 11 (IL-11 ) POLYPEPTIDES

[0033] Aspects of the present disclosure include interleukin 11 (IL-1 1 ) polypeptides. In some embodiments, an IL-1 1 polypeptide of the present disclosure comprises one or more amino acid substitutions which increase affinity of the IL-1 1 polypeptide for human IL-1 1 receptor (IL-11 R) as compared to wild-type human IL-1 1 . Alternatively, or additionally, an IL-1 1 polypeptide of the present disclosure may comprise one or more amino acid substitutions which inhibit formation of a complex comprising the IL-1 1 polypeptide, human IL-1 1 R and glycoprotein 130 (gp130) upon binding of the IL-1 1 polypeptide to human IL-11 R. The present disclosure is based in part on the development of high affinity IL-1 1 variants in which binding to the gp130 co-receptor is ablated, thereby providing high affinity IL-11 R antagonists capable of disrupting IL-1 1 / IL-1 1 R signaling and, e.g., tumor progression as demonstrated in the Experimental section below. Details regarding embodiments of the IL-11 polypeptides will now be described.

[0034] The terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

[0035] The term “amino acid” generally refers to any monomer unit that comprises a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, e.g., thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising photoactivatable cross-linkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids, other carbohydrate modified amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox- active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moieties.

[0036] The term “amino acid” includes, but is not limited to, naturally-occurring a-amino acids and their stereoisomers. “Stereoisomers” of amino acids refer to mirror image isomers of the amino acids, such as L-amino acids or D-amino acids. For example, a stereoisomer of a naturally-occurring amino acid refers to the mirror image isomer of the naturally-occurring amino acid ( / .e., the D-amino acid).

[0037] Naturally-occurring a-amino acids are those encoded by the genetic code as well as those amino acids that are later modified (e.g., hydroxyproline, y-carboxyglutamate, and 0- phosphoserine). Naturally-occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally- occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D- aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D- isoleucine (D-lle), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-GIn), D-serine (D-Ser), D-threonine (D- Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0038] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. For example, an L-amino acid may be represented herein by its commonly known three letter symbol (e.g., Arg for L-arginine) or by an upper-case one-letter amino acid symbol (e.g., R for L-arginine). A D-amino acid may be represented herein by its commonly known three letter symbol (e.g., D-Arg for D-arginine) or by a lower-case one-letter amino acid symbol (e.g., r for D-arginine).

[0039] An IL-1 1 polypeptide of the present disclosure may comprise one or more amino acid substitutions which increase affinity of the IL-1 1 polypeptide for human IL-1 1 R as compared to wild-type human IL-11 . As will be appreciated with the benefit of the present disclosure, such IL- 1 1 polypeptides find use as high affinity IL-1 1 R agonists when signaling through IL-1 1 is desired, or alternatively, as high affinity IL-1 1 R antagonists (e.g., when the IL-1 1 polypeptide is further engineered to inhibit IL-11 / IL-11 R / gp130 complex formation) when disruption of IL-1 1 signaling is desired. In some instances, the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-1 1 R comprise a substitution at D19, T21 , L57, G62, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1 . According to some embodiments, the one or more amino acid substitutions which increase affinity of the IL-1 1 polypeptide for human IL-11 R comprise D G, T211, L57P, G62R, or any combination thereof. In some instances, the IL-11 polypeptide comprises one, two, three or each of the D19, T21 , L57, and G62 substitutions. According to some embodiments, the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-1 1 R comprise a substitution at G71 , V72, or both. Non-limiting examples of such substitutions include G71 D, V72M, or both.

[0040] In certain embodiments, the one or more amino acid substitutions which increase affinity of the IL-1 1 polypeptide for human IL-1 1 R comprise D19G and optionally one or more of T211, L57P, G62R, G71 D, and / or V72M. In some instances, the one or more amino acid substitutions which increase affinity of the IL-1 1 polypeptide for human IL-1 1 R comprise T21 I and optionally one or more of D19G, L57P, G62R, G71 D, and / or V72M. According to some embodiments, the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 R comprise L57P and optionally one or more of D19G, T21 1, G62R, G71 D, and / or V72M. In certain embodiments, the one or more amino acid substitutions which increase affinity of the IL- 1 1 polypeptide for human IL-11 R comprise G62R and optionally one or more of D19G, T21 1, L57P, G71 D, and / or V72M.

[0041] The binding affinity of an IL-1 1 polypeptide for IL-11 R can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 or BIAcore T200 instrument, using general procedures outlined by the manufacturer); by Bio-Layer Interferometry (BLI) (e.g., using the Octet(R)platform by Sartorius, Inc.); by radioimmunoassay; or the like. A suitable IL-1 1 R polypeptide that may be employed to assess the binding affinity of an IL-1 1 polypeptide for human IL-11 R is the follosing human IL-1 1 Ra polypeptide: UniProt Q14626 - I11 RA_HUMAN. A suitable IL-1 1 R polypeptide that may be employed to assess the binding affinity of an IL-1 1 polypeptide for mouse IL-1 1 R is the following mouse IL-1 1 Ra polypeptide: UniProt Q64385 - 11 1 RA_MOUSE.

[0042] An IL-1 1 polypeptide of the present disclosure may comprise one or more amino acid substitutions which inhibit formation of a complex comprising the IL-11 polypeptide, human IL- U R and gp130 upon binding of the IL-11 polypeptide to human IL-11 R. As will be appreciated with the benefit of the present disclosure, such IL-11 polypeptides find use as IL-1 1 R antagonists. In some instances, the one or more amino acid substitutions which inhibit formation of a complex comprising the IL-1 1 polypeptide, human IL-1 1 R and gp130 upon binding of the IL-11 polypeptide to human IL-1 1 R comprise a substitution at L24, W147, or both, wherein numbering of positions is according to SEQ ID NO: 1. According to some embodiments, the one or more amino acid substitutions comprise L24R, W147A, or both. In one non-limiting example, the one or more amino acid substitutions comprise L24R and W147A.

[0043] In some instances, an IL-1 1 polypeptide of the present disclosure cross-reacts with a nonhuman IL-11 R. A polypeptide of the present disclosure is said to be "cross- reactive" for two different antigens or antigenic determinants (e.g., IL-11 R from two different species of mammal, such as human and mouse, or human and cynomolgus monkey) if it is specific for (as defined herein) both these different antigens or antigenic determinants. In certain embodiments, an IL-1 1 polypeptide binding to antigen 1 (Ag1 ) is "cross-reactive" to antigen 2 (Ag2) when the EC50 and / or KDvalues are in a similar range for both antigens. According to some embodiments, an IL-1 1 polypeptide binding to Ag1 is cross-reactive to Ag2 when the ratio of affinity for Ag1 to affinity for Ag2 is equal or less 10 (< 10) and equal or greater than 0.1 (>0.1 ), which means that the affinities for Ag1 and Ag2 do not differ more than a factor of 10 (the affinities are within one order of magnitude of monovalent KD), on condition that affinities are measured with the same method in the same experimental setting for both antigens. Accordingly, an IL-1 1 polypeptide of the present disclosure may have a ratio of affinity for human IL-1 1 R to the affinity for mouse IL-11 R which is equal or less 10 (< 10) and equal or greater than 0.1 (>0.1 ), which means that the affinities for human and mouse IL-11 R do not differ more than a factor of 10 (the affinities are within one order of magnitude of monovalent KD). Such an IL-1 1 polypeptide may be used, e.g., in toxicological and / or efficacy studies performed in mouse because the toxicity profile observed in mouse would be relevant to anticipate potential adverse effects in humans.

[0044] Accordingly, in some embodiments, an IL-1 1 polypeptide of the present disclosure crossreacts with a non-human animal IL-11 R. In certain embodiments, the non-human animal IL-1 1 R is mouse IL-11 R. For example, an IL-11 polypeptide of the present disclosure may comprise one or more amino acid substitutions which increase affinity of the IL-1 1 polypeptide for mouse IL- U R as compared to wild-type human IL-1 1 . In some instances, the one or more amino acid substitutions which increase affinity of the IL-1 1 polypeptide for mouse IL-1 1 R comprise a substitution at D41 , S60, D79, H86, G158, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1. Non-limiting examples of such substitutions comprise D41 E, S60T, D79Y, H86Y, G158E, or any combination thereof. In certain embodiments, the one or more amino acid substitutions which increase affinity of the IL-1 1 polypeptide for mouse IL- 1 1 R comprise S60T.

[0045] Amino acid substitutions of interest may be generated using a variety of approaches known to those of ordinary skill in the art. A non-limiting example approach for producing variant polypeptides comprising one or more amino acid substitutions of interest is site-directed mutagenesis (SDM). SDM is an in vitro procedure that uses custom designed oligonucleotide primers to confer a desired mutation in a double-stranded DNA plasmid. The most widely-used methods do not require any specific modifications or unique E coli strains and incorporate mutations into the plasmid by inverse PCR with standard primers. For these methods, primers can be designed in either an overlapping or a back-to-back orientation. Overlapping primer design results in a product that will re-circularize to form a doubly-nicked plasmid. Despite the presence of these nicks, this circular product can be directly transformed into E. coli, albeit at a lower efficiency than non-nicked plasmids. Back-to-back primer design methods not only have the advantage of transforming non-nicked plasmids, but also allow exponential amplification, generating significantly more of the desired product. In addition, because the primers do not overlap each other, deletion sizes are only limited by the plasmid and insertions are only limited by the constraints of modern primer synthesis. Currently, by splitting the insertion between the two primers, insertions up to 100 bp can routinely be created in one step using this method. Tools and kits for primer design and performing SDM are readily available and include, e.g., the NEBaseChanger primer design tool and the Q5 Site-Directed Mutagenesis Kit available from New England Biolabs.

[0046] Table 1 - Wild-type Human IL-11 Amino Acid Sequence

[0047] The present disclosure provides the polypeptide provided above (SED ID NO: 1 ) comprising one or more of any of the amino acid substitutions described elsewhere herein in any combination, as well as nucleic acids that encode such polypeptides. Cells comprising such polypeptides and nucleic acids are also provided. As will be appreciated, the present disclosure also provides variants of any of the polypeptides of the present disclosure, where in some instances a variant polypeptide comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to the parental / reference sequence, or a functional fragment thereof, where the variant retains the functionality (e.g., increased affinity for human IL- 1 1 R; inhibition of formation of a complex comprising the IL-11 polypeptide, human IL-11 R and gp130 upon binding of the IL-1 1 polypeptide to human IL-1 1 R; cross-reactivity with mouse IL- 1 1 R, and / or the like) of the parental / reference sequence.

[0048] In certain embodiments, in addition to the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-1 1 R, the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for mouse IL-11 R, and / or the one or more amino acid substitutions which inhibit formation of a complex comprising the IL-1 1 polypeptide, human IL-1 1 R and gp130 upon binding of the IL-1 1 polypeptide to human IL-11 R, an IL-1 1 polypeptide of the present disclosure may further comprise one or more additional amino acid substitutions, e.g., conservative or non-conservative substitutions. Conservative substitutions are shown in the table below (right-most column) More substantial changes are provided in the table below under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an IL-1 1 polypeptide of interest and the products screened for a desired activity, e.g., retained or improved increase in affinity for human IL-1 1 R; retained or improved inhibition of formation of a complex comprising the IL-1 1 polypeptide, human IL-11 R and gp130 upon binding of the IL-11 polypeptide to human IL-11 R; retained or improved cross- reactivity with mouse IL-11 R, and / or the like.

[0049] Amino Acid Substitutions Amino acids may be grouped according to common side-chain properties:

[0050] (1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie;

[0051] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0052] (3) acidic: Asp, Glu;

[0053] (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro;

[0054] (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0055] In certain embodiments, the IL-11 polypeptide is conjugated to an agent. The term “conjugated” generally refers to a chemical linkage, either covalent or non-covalent, usually covalent, that proximally associates one molecule of interest with a second molecule of interest. In certain embodiments, the agent conjugated to the IL-11 polypeptide is selected from a chemotherapeutic agent, a toxin, a radiation-sensitizing agent, a radioactive isotope (e.g., a therapeutic radioactive isotope), a detectable label, and a half-life extending moiety.

[0056] According to some embodiments, the agent is a therapeutic agent, e.g., a chemotherapeutic agent. Therapeutic agents of interest include agents capable of affecting the function of a cell / tissue to which the conjugate binds via specific binding of the IL-11 polypeptide to IL-11 R. When the function of the cell / tissue is pathological, an agent that reduces the function of the cell / tissue may be employed. In certain aspects, a conjugate of the present disclosure includes an agent that reduces the function of a target cell / tissue by inhibiting cell proliferation and / or killing the cell / tissue. Such agents may vary and include cytostatic agents and cytotoxic agents, e.g., an agent capable of killing a target cell tissue with or without being internalized into a target cell.

[0057] In certain embodiments, the therapeutic agent is a cytotoxic agent selected from an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. In some embodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065, CPT-11 (SN-38), topotecan, doxorubicin, morpholino-doxorubicin, rhizoxin, cyanomorpholinodoxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, maytansine DM1 , maytansine DM4, DM-1 , an auristatin or other dolastatin derivatives, such as auristatin E or auristatin F, AEB (AEB-071), AEVB (5-benzoylvaleric acid-AE ester), AEFP (antibody- endostatin fusion protein), MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), pyrrolobenzodiazepines (PBDs) , eleutherobin, netropsin, or any combination thereof.

[0058] According to some embodiments, the agent is a toxin, such as a protein toxin selected from hemiasterlin and hemiasterlin analogs such as HTI-286 (e.g., see USPN 7,579,323; WO 2004 / 026293; and USPN 8,129,407, the full disclosures of which are incorporated herein by reference), abrin, brucine, cicutoxin, diphtheria toxin, batrachotoxin, botulism toxin, shiga toxin, endotoxin, Pseudomonas exotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin, falcarinol, fumonisin Bl, fumonisin B2, afla toxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcicludine, geldanamycin, gelonin, lotaustralin, ocratoxin A, patulin, ricin, strychnine, trichothecene, zearlenone, and tetradotoxin. Enzymatically active toxins and fragments thereof which may be employed include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.

[0059] In certain embodiments, the agent is a radiation-sensitizing agent. As used herein, a “radiation-sensitizing agent” is an agent that enhances the ability of radiation to kill tumor cells. Non-limiting examples of radiation-sensitizing agents that may be conjugated to the IL-1 1 polypeptide include cisplatin, 5-fluorouracil (5-FU), AZD7762, selumetinib, and the like.

[0060] In certain embodiments, the agent is a radioisotope, e.g., useful for therapy and / or detection (e.g., imaging). Non-limiting examples of radioisotopes that may be conjugated to the IL-11 polypeptide include but are not limited to225Ac,111Ag,114Ag,71As,72As,77As,211At,198Au,199Au,212Bi,213Bi,75Br,76Br,11C,13C,55Co,62Cu,64Cu,67Cu,165Dy,166Dy,169Er,18F,19F,52Fe,59Fe,S6Ga,67Ga,68Ga,72Ga,154 158Gd,157Gd,159Gd,166Ho,120l,121l,123l,124l,125l,131l,110ln,111In,113mln,

[0061] 121Sn,83Sr,89Sr,161Tb,94Tc, "Tc,99mTc,227Th,201Tl,172Tm,127Te,90Y,169Yb,175Yb,133X, and89Zr

[0062] In certain embodiments, a radioisotope is conjugated to the IL-1 1 polypeptide via a chelator, for example, a bifunctional chelator. A bifunctional chelator may contain a metal chelating moiety that binds the radioisotope in a stable coordination complex and a reactive functional group that is covalently linked to the IL-11 polypeptide, so that the radioisotope may be properly directed to the desirable molecular target in vivo. Non-limiting examples of bifunctional chelators that may be employed to conjugate an IL-11 polypeptide of the present disclosure to a radioisotope include p-SCN-Bn-DOTA and p-SCN-Bn-deferoxamine. Additional examples of bifunctional chelators that may be employed to conjugate an IL-1 1 polypeptide of the present disclosure to a radioisotope include those described in Price & Orvig (2014) Chem. Soc. Rev. 43:260; and Brechbiel (2008) Q J Nucl Med Mol Imaging 52(2):166-173.

[0063] According to some embodiments, the radioisotope is a therapeutic radioisotope. In certain embodiments, the radioisotope is an alpha emitting radioisotope, e.g.,225Ac,211At,212Bi / 212Pb,213Bi,223Ra, or227Th. In other embodiments, the radioisotope is a beta minus emitting radioisotope, e.g.,32P,33P,67Cu,90Y,1311 or177Lu.

[0064] According to some embodiments, the agent is a labeling agent. By “labeling agent” (or “detectable label”) is meant the agent detectably labels the IL-1 1 polypeptide, such that the IL-1 1 polypeptide may be detected in an application of interest (e.g., in vitro and / or in vivo research and / or clinical applications). Detectable labels of interest include radioisotopes (e.g., gamma or positron emitters), enzymes that generate a detectable product (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, etc.), fluorescent proteins, paramagnetic atoms, and the like. In certain aspects, the IL-1 1 polypeptide is conjugated to a specific binding partner of detectable label, e.g., conjugated to biotin such that detection may occur via a detectable label that includes avidin / streptavidin.

[0065] In certain embodiments, the agent is a labeling agent that finds use in in vivo imaging, such as near-infrared (NIR) optical imaging, single-photon emission computed tomography (SPECT) ± CT imaging, positron emission tomography (PET) ± CT imaging, nuclear magnetic resonance (NMR) spectroscopy, or the like. Labeling agents that find use in such applications include, but are not limited to, fluorescent labels, radioisotopes, and the like. In certain aspects, the labeling agent is a multi-modal in vivo imaging agent that permits in vivo imaging using two or more imaging approaches (e.g., see Thorp-Greenwood and Coogan (2011 ) Dalton Trans. 40:6129-6143).

[0066] In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in near-infrared (NIR) imaging applications. Such agents include, but are not limited to, a Kodak X- SIGHT dye, Pz 247, DyLight 750 and 800 Fluors, Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, IRDye 680 and 800CW Fluors. According to some embodiments, the labeling agent is an in vivo imaging agent that finds use in SPECT imaging applications, non-limiting examples of which include99mTc,111ln,123l,201TI, and133Xe. In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in PET imaging applications, e.g.,11C,13N,15O,18F,64Cu,S2Cu,124l,76Br,82Rb,S8Ga, or the like.

[0067] For half-life extension, the IL-1 1 polypeptide may be conjugated to an agent that provides for an improved pharmacokinetic profile (e.g., by PEGylation, hyperglycosylation, and the like). Modifications that can enhance serum half-life are of interest. A subject IL-1 1 polypeptide may be “PEGylated”, as containing one or more polyethylene glycol) (PEG) moieties. Methods and reagents suitable for PEGylation of a protein are well known in the art and may be found, e.g., in US Pat. No. 5,849,860. PEG suitable for conjugation to a protein is generally soluble in water at room temperature and has the general formula R(O-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. Where R is a protective group, it generally has from 1 to 8 carbons. The PEG conjugated to the subject IL-11 polypeptide can be linear. The PEG conjugated to the IL-11 polypeptide may also be branched. Branched PEG derivatives such as those described in U.S. Pat. No. 5,643,575, “star-PEGs” and multi-armed PEGs. Star PEGs are described in the art including, e.g., in U.S. Patent No. 6,046,305.

[0068] Any of the above agents may be conjugated to the IL-1 1 polypeptide via a linker. If present, the linker molecule(s) may be of sufficient length to permit the IL-11 polypeptide and the linked agent to allow some flexible movement between the IL-1 1 polypeptide and the linked agent. Linker molecules may be, e.g., about 6-50 atoms long. Linker molecules may also be, e.g., aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Where the linkers are peptides, the linkers can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 or more amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1 , 2, 3, 4, 5, 6, or 7 amino acids in length.

[0069] Flexible linkers include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used where relatively unstructured amino acids are of interest, and may serve as a neutral tether between components. The ordinarily skilled artisan will recognize that design of an IL-11 polypeptide conjugated to any agents described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer a less flexible structure.

[0070] According to some embodiments, the IL-11 polypeptide is conjugated to the agent via a non-cleavable linker. Non-cleavable linkers of interest include, but are not limited to, thioether linkers. An example of a thioether linker that may be employed includes a succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) linker.

[0071] Numerous strategies are available for linking agents to an IL-11 polypeptide directly, or indirectly via a linker. For example, the agent may be derivatized by covalently attaching a linker to the agent, where the linker has a functional group capable of reacting with a “chemical handle” on the IL-1 1 polypeptide. The functional group on the linker may vary and may be selected based on compatibility with the chemical handle on the IL-11 polypeptide. According to one embodiment, the chemical handle on the IL-1 1 polypeptide is provided by incorporation of an unnatural amino acid having the chemical handle into the IL-11 polypeptide. Unnatural amino acids which find use for preparing the conjugates of the present disclosure include those having a functional group selected from an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde (e.g., formylglycine, e.g., SMARTag™ technology from Catalent Pharma Solutions), nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, and boronic acid functional group. Unnatural amino acids which may be incorporated into an IL-11 polypeptide of a conjugate of the present disclosure, which unnatural amino acid may be selected to provide a functional group of interest, are known and described in, e.g., Maza et al. (2015) Bioconjug. Chem. 26(9):1884-9; Patterson et al. (2014) ACS Chem. Biol. 9:592-605; Adumeau et al. (2016) Mol. Imaging Biol. (2):153-65; and elsewhere. An unnatural amino acid may be incorporated into an IL-1 1 polypeptide via chemical synthesis or recombinant approaches, e.g., using a suitable orthogonal amino acyl tRNA synthetase-tRNA pair for incorporation of the unnatural amino acid during translation of the IL-11 polypeptide in a host cell.

[0072] The functional group of an unnatural amino acid present in the IL-1 1 polypeptide may be an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde, asaldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, boronic acid, diazo, tetrazine, tetrazole, quadrocyclane, iodobenzene, or other suitable functional group, and the functional group on the linker is selected to react with the functional group of the unnatural amino acid (or vice versa). As just one example, an azide-bearing unnatural amino acid (e.g., 5-azido-L-norvaline, or the like) may be incorporated into the IL-1 1 polypeptide and the linker portion of a linker-agent moiety may include an alkyne functional group, such that the IL-1 1 polypeptide and linker-agent moiety are covalently conjugated via azide-alkyne cycloaddition. Conjugation may be carried out using, e.g., a copper-catalyzed azide-alkyne cycloaddition reaction.

[0073] In certain embodiments, the chemical handle on the IL-1 1 polypeptide does not involve an unnatural amino acid. An IL-1 1 polypeptide containing no unnatural amino acids may be conjugated to the agent by utilizing, e.g., nucleophilic functional groups of the IL-11 polypeptide (such as the N-terminal amine or the primary amine of lysine, or any other nucleophilic amino acid residue) as a nucleophile in a substitution reaction with a moiety bearing a reactive leaving group or other electrophilic group. An example would be to prepare an agent-linker moiety bearing an N-hydroxysuccinimidyl (NHS) ester and allow it to react with the IL-11 polypeptide under aqueous conditions at elevated pH (~10) or in polar organic solvents such as DMSO with an added non-nucleophilic base, such as N,N-diisopropylethylamine.

[0074] It will be appreciated that the particular approach for attaching a linker, agent and / or IL- 1 1 polypeptide to each other may vary depending upon the particular linker, agent and / or IL-1 1 polypeptide and functional groups selected and employed for conjugating the various components to each other.

[0075] Methods of Producing IL-1 1 Polypeptides

[0076] Using the information provided herein, the IL-1 1 polypeptides of the present disclosure may be prepared using standard techniques well known to those of skill in the art. For example, a nucleic acid sequence(s) encoding the amino acid sequence of an IL-1 1 polypeptide of the present disclosure can be used to express the IL-1 1 polypeptide. The polypeptide sequences provided herein (see, e.g., Table 1 ) can be used to determine appropriate nucleic acid sequences encoding the IL-11 polypeptides and the nucleic acids sequences then used to express one or more IL-11 polypeptides. The nucleic acid sequence(s) can be optimized to reflect particular codon “preferences” for various expression systems according to standard methods well known to those of skill in the art. Using the sequence information provided, the nucleic acids may be synthesized according to a number of standard methods known to those of skill in the art.

[0077] Once a nucleic acid(s) encoding a subject IL-11 polypeptide is synthesized, it can be amplified and / or cloned according to standard methods. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are known to persons of skill in the art and are the subjects of numerous textbooks and laboratory manuals. Expression of natural or synthetic nucleic acids encoding the IL-1 1 polypeptides of the present disclosure can be achieved by operably linking a nucleic acid encoding an IL-1 1 polypeptide to a promoter (which is either constitutive or inducible), and incorporating the construct into an expression vector to generate a recombinant expression vector. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the IL-1 1 polypeptide. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0078] To obtain high levels of expression of a cloned nucleic acid it is common to construct expression plasmids which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription / translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) operon. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing IL-11 polypeptides are available using, for example, E. coli, Bacillus sp. and Salmonella. E. coli systems may also be used.

[0079] A nucleic acid encoding an IL-1 1 polypeptide of the present disclosure may also be subcloned into an expression vector that allows for the addition of a tag (e.g., FLAG, his (e.g., hexahistidine), and the like) at the C-terminal end or the N-terminal end of the IL-11 polypeptide to facilitate purification. Methods of transfecting and expressing genes in mammalian cells are known in the art. Transducing cells with nucleic acids can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells within the host range of the vector. The culture of cells used in the present disclosure, including cell lines and cultured cells from tissue (e.g., tumor) or blood samples is well known in the art.

[0080] Once the nucleic acid encoding a subject IL-11 polypeptide is isolated and cloned, one can express the nucleic acid in a variety of recombinantly engineered cells known to those of skill in the art. Examples of such cells include bacteria, yeast, filamentous fungi, insect (e.g., those employing baculoviral vectors), and mammalian cells.

[0081] Isolation and purification of a subject IL-1 1 polypeptide can be accomplished according to methods known in the art. For example, a protein can be isolated from a lysate of cells genetically modified to express the protein constitutively and / or upon induction, or from a synthetic reaction mixture, by immunoaffinity purification (or precipitation using Protein L or A), washing to remove non-specifically bound material, and eluting the specifically bound IL-1 1 polypeptide. The isolated IL-1 1 polypeptide can be further purified by dialysis and other methods normally employed in protein purification methods. In one embodiment, the IL-1 1 polypeptide may be isolated using metal chelate chromatography methods. IL-11 polypeptides of the present disclosure may contain modifications to facilitate isolation, as discussed elsewhere herein.

[0082] The IL-11 polypeptides may be prepared in substantially pure or isolated form (e.g., free from other polypeptides). The protein can be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). Purified IL-11 polypeptides may be provided such that the IL-1 1 polypeptide is present in a composition that is substantially free of other expressed proteins, e.g., less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed proteins.

[0083] The IL-11 polypeptides produced by prokaryotic cells may require exposure to chaotropic agents for proper folding. During purification from E. coli, for example, the expressed protein can be optionally denatured and then renatured. This can be accomplished, e.g., by solubilizing the bacterially produced IL-11 polypeptides in a chaotropic agent such as guanidine HCI. The IL-1 1 polypeptide is then renatured, either by slow dialysis or by gel filtration. Alternatively, nucleic acid encoding the IL-11 polypeptide may be operably linked to a secretion signal sequence such as pelB so that the IL-1 1 polypeptide is secreted into the periplasm in correctly-folded form.

[0084] The present disclosure also provides cells that produce the IL-1 1 polypeptides of the present disclosure, where suitable cells include eukaryotic cells (e.g., mammalian cells) and prokaryotic cells, e.g., bacterial cells. When bacterial cells are employed, in some embodiments, endotoxin is removed from the IL-1 1 polypeptide subsequent to expression, and / or the bacterial cells are genetically modified such that they do not produce endotoxin. The present disclosure provides a recombinant host cell (also referred to herein as a “genetically modified host cell”) that is genetically modified with a nucleic acid comprising a nucleotide sequence encoding an IL-1 1 polypeptide of the present disclosure. Non-limiting examples of recombinant host cells include Chinese Hamster Ovary (CHO) cells (e.g., CHO-S, CHO-K1 , and CHO-DG44), HEK293 cells, NSO cells, and the like.

[0085] NUCLEIC ACIDS, EXPRESSION VECTORS AND CELLS

[0086] In view of the section above regarding methods of producing the IL-1 1 polypeptides of the present disclosure, it will be appreciated that the present disclosure also provides nucleic acids, expression vectors and cells.

[0087] According to some embodiments, provided is a nucleic acid encoding an IL-1 1 polypeptide of the present disclosure. Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of an IL-1 1 polypeptide of the present disclosure provides a description of all the polynucleotides capable of encoding the IL- 1 1 polypeptide. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the IL-11 polypeptides disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the IL-1 1 polypeptide of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any IL-1 1 polypeptide disclosed herein.

[0088] Also provided are expression vectors comprising any of the nucleic acids of the present disclosure. Expression of natural or synthetic nucleic acids encoding the IL-11 polypeptides of the present disclosure can be achieved by operably linking a nucleic acid encoding the IL-1 1 polypeptide to a promoter (which is either constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the IL-1 1 polypeptide. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0089] Cells that comprise any of the nucleic acids and / or expression vectors of the present disclosure are also provided. Also provided are methods of making a IL-1 1 polypeptide of the present disclosure, the methods including culturing a cell of the present disclosure under conditions suitable for the cell to express the IL-11 polypeptide, where the IL-1 1 polypeptide is produced. The conditions for culturing the cell such that the IL-1 1 polypeptide is expressed may vary. Such conditions may include culturing the cell in a suitable container (e.g., a cell culture plate or well thereof), in suitable medium (e.g., cell culture medium, such as DMEM, RPMI, MEM, IMDM, DMEM / F-12, or the like) at a suitable temperature (e.g., 32°C - 42°C, such as 37°C) and pH (e.g., pH 7.0 - 7.7, such as pH 7.4) in an environment having a suitable percentage of CO2, e.g., 3% to 10%, such as 5%).

[0090] COMPOSITIONS

[0091] Aspects of the present disclosure further include compositions. According to some embodiments, a composition of the present disclosure includes a IL-11 polypeptide. For example, the IL-1 1 polypeptide may be any of the IL-1 1 polypeptides described in the IL-1 1 Polypeptides section hereinabove and / or in the Experimental section below, which descriptions are incorporated but not reiterated herein for purposes of brevity.

[0092] In certain aspects, a composition of the present disclosure includes the IL-11 polypeptide present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCh, KCI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl- 3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, a protease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

[0093] A composition of the present disclosure may be formulated for administration to a subject (e.g., a human subject) in need thereof. Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, a pharmaceutical composition of the present disclosure includes an IL-11 polypeptide of the present disclosure, and a pharmaceutically acceptable carrier.

[0094] The IL-1 1 polypeptide can be incorporated into a variety of formulations for therapeutic administration. More particularly, the IL-1 1 polypeptides can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.

[0095] Formulations of the IL-11 polypeptides for administration to a subject (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0096] In pharmaceutical dosage forms, the IL-1 1 polypeptides can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0097] For oral preparations, the IL-1 1 polypeptides can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0098] The IL-11 polypeptides can be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration. In certain embodiments, the IL-11 polypeptides are formulated for injection by dissolving, suspending or emulsifying the IL-11 polypeptides in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0099] Pharmaceutical compositions that include the IL-1 1 polypeptides may be prepared by mixing the IL-11 polypeptides having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof) ; amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0100] The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration.

[0101] An aqueous formulation of the IL-11 polypeptides may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation. A tonicity agent may be included to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0102] A surfactant may also be added to the formulation to reduce aggregation and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1 % w / v.

[0103] A lyoprotectant may also be added in order to protect the IL-11 polypeptides against destabilizing conditions during a lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included, e.g., in an amount of about 10 mM to 500 nM.

[0104] In some embodiments, a pharmaceutical composition includes the IL-1 1 polypeptide, and one or more of the above-identified components (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% weight / volume (w / v).

[0105] METHODS OF USE

[0106] Aspects of the present disclosure further include methods of using the IL-1 1 polypeptides of the present disclosure. For example, in certain embodiments, provided are methods of treating a condition associated with IL-1 1 R signaling in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising an IL-11 polypeptide of the present disclosure. IL-11 / IL-11 R signaling has been implicated in a variety of diseases across tissue types.9Increased IL-1 1 signaling has been associated with cancer proliferation, metastasis, and chemoresistance,14 16leading to the progression of gastric, colorectal, breast, and bone cancers among others.19 22IL-11 has also been implicated in fibrotic remodeling that affects cancer associated fibroblasts as well as lung, liver, and heart fibrosis.1823 26

[0107] In certain embodiments, the condition associated with IL-1 1 R signaling is a cell proliferative disorder. By “cell proliferative disorder” is meant a disorder wherein unwanted cell proliferation of one or more subset(s) of cells in a multicellular organism occurs, resulting in harm, for example, pain or decreased life expectancy to the organism. Cell proliferative disorders include, but are not limited to, cancer, pre-cancer, benign tumors, blood vessel proliferative disorders (e.g., arthritis, restenosis, and the like), fibrotic disorders (e.g., fibrosis of the lung, liver, heart, and the like), psoriasis, epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteoplastic tumors, dysplastic masses, mesangial cell proliferative disorders, and the like.

[0108] In some embodiments, the condition associated with IL-11 R signaling is cancer. The subject methods may be employed for the treatment of a large variety of cancers. “Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancers that may be treated using the subject methods include, but are not limited to, carcinoma, lymphoma, blastoma, and sarcoma. In some instances, when the condition associated with IL-1 1 R signaling is cancer, the cancer is a lung cancer (e.g., lung adenocarcinoma), gastric cancer, colorectal cancer, breast cancer, or bone cancer. In some instances, the condition is associated with IL-1 1 R signaling by cancer- associated fibroblasts (CAFs).

[0109] More particular examples of cancers which may be treated according to the methods of the present disclosure include renal cancer; kidney cancer; glioblastoma multiforme; metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced bone osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease) and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; cervical carcinoma; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyclic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; colorectal cancer, KRAS mutated colorectal cancer; colon carcinoma; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancers such as KRAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; lung carcinoma; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, androgen-independent prostate cancer, androgendependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or uterer); renal carcinoma; Wilms' tumor; and bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In some embodiments, the cancer is myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, or papillary adenocarcinomas.

[0110] In some embodiments, the condition associated with IL-1 1 R signaling is fibrosis. Fibrosis is characterized by the overgrowth, hardening, and / or scarring of tissue and is attributed to excess deposition of extracellular matrix components including collagen. IL-11 R signaling has been implicated in various types of fibrosis including fibrosis of the lung, liver, and heart.2528

[0111] The IL-1 1 polypeptides of the present disclosure may be administered via a route of administration selected from oral (e.g., in tablet form, capsule form, liquid form, or the like), parenteral (e.g., by intravenous, intra-arterial, subcutaneous, intramuscular, epidural injection), topical, intra-nasal, intra-tumoral administration, or intraperitoneal (IP) administration.

[0112] The IL-1 1 polypeptides of the present disclosure may be administered (e.g., in a pharmaceutical composition) in a therapeutically effective amount. By “therapeutically effective amount” is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a cancer, a reduction in or slowing of fibrosis, and / or the like, as compared to a control. With respect to cancer, in some embodiments, the therapeutically effective amount is sufficient to slow the growth of a tumor, reduce the size of a tumor, and / or the like. An effective amount can be administered in one or more administrations.

[0113] Provided are methods of treating a condition associated with IL-1 1 R signaling in a subject in need thereof. By treatment is meant at least an amelioration of one or more symptoms associated with the condition associated with IL-1 1 R signaling of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition associated with IL-11 R signaling being treated. As such, treatment also includes situations where the condition associated with IL-11 R signaling, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the individual no longer suffers from the condition associated with IL-1 1 R signaling, or at least the symptoms that characterize the condition associated with IL-1 1 R signaling.

[0114] An IL-1 1 polypeptide of the present disclosure may be administered to the individual alone or in combination with a second agent. Second agents of interest include, but are not limited to, agents approved by the United States Food and Drug Administration and / or the European Medicines Agency (EMA) for use in treating cancer, or for use in treating fibrosis. In some embodiments, the second agent is a chemotherapeutic agent. Non-limiting examples of such chemotherapeutic agents include Capecitabine, Carboplatin, Fluorouracil, Busulfan, Cyclophosphamide, Daunorubicin, Gemcitabine, Altretamine, Fludarabine, Cytarabine, Docetaxel, Etoposide, Mercaptopurine, Methotrexate, Alkylating antineoplastic agent, Azacitidine, Carmustine, Chlorambucil, Dacarbazine, Floxuridine, Melphalan, Paclitaxel, Pentostatin, Temozolomide, or any combination thereof. In some embodiments, the second agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors of interest include, but are not limited to, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed cell death-1 (PD-1 ) inhibitor, a programmed cell death ligand-1 (PD-L1 ) inhibitor, a lymphocyte activation gene-3 (LAG-3) inhibitor, a T-cell immunoglobulin domain and mucin domain 3 (TIM- 3) inhibitor, an indoleamine (2,3)-dioxygenase (IDO) inhibitor, a T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitor, a V-domain Ig suppressor of T cell activation (VISTA) inhibitor, a B7-H3 inhibitor, and any combination thereof.

[0115] When an IL-1 1 polypeptide of the present disclosure is administered with a second agent, the IL-11 polypeptide and the second agent may be administered to the individual according to any suitable administration regimen. According to certain embodiments, the IL-11 polypeptide and the second agent are administered according to a dosing regimen approved for individual use. In some embodiments, the administration of the IL-1 1 polypeptide permits the second agent to be administered according to a dosing regimen that involves one or more lower and / or less frequent doses, and / or a reduced number of cycles as compared with that utilized when the second agent is administered without administration of the IL-1 1 polypeptide. In certain aspects, the administration of the second agent permits the IL-1 1 polypeptide to be administered according to a dosing regimen that involves one or more lower and / or less frequent doses, and / or a reduced number of cycles as compared with that utilized when the IL-1 1 polypeptide is administered without administration of the second agent.

[0116] In some embodiments, one or more doses of the IL-1 1 polypeptide and the second agent are administered concurrently to the individual. By “concurrently” is meant the IL-11 polypeptide and the second agent are either present in the same pharmaceutical composition, or the IL-1 1 polypeptide and the second agent are administered as separate pharmaceutical compositions within 1 hour or less, 30 minutes or less, or 15 minutes or less.

[0117] In some embodiments, one or more doses of the IL-1 1 polypeptide and the second agent are administered sequentially to the individual.

[0118] In some embodiments, the IL-1 1 polypeptide and the second agent are administered to the individual in different compositions and / or at different times. For example, the IL-1 1 polypeptide may be administered prior to administration of the second agent, e.g., in a particular cycle. Alternatively, the second agent may be administered prior to administration of the IL-1 1 polypeptide, e.g., in a particular cycle. The second agent to be administered may be administered a period of time that starts at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or up to 5 days or more after the administration of the first agent to be administered.

[0119] In one example, the second agent is administered to the individual for a desirable period of time prior to administration of the IL-1 1 polypeptide. In certain aspects, when the individual has cancer, such a regimen “primes” the cancer cells to potentiate the anti-cancer effect of the IL-11 polypeptide. Such a period of time separating a step of administering the second agent from a step of administering the IL-11 polypeptide is of sufficient length to permit priming of the cancer cells, desirably so that the anti-cancer effect of the IL-1 1 polypeptide is increased.

[0120] In some embodiments, administration of one agent is specifically timed relative to administration of the other agent. For example, in some embodiments, the IL-1 1 polypeptide is administered so that a particular effect is observed (or expected to be observed, for example based on population studies showing a correlation between a given dosing regimen and the particular effect of interest).

[0121] In certain aspects, desired relative dosing regimens for agents administered in combination may be assessed or determined empirically, for example using ex vivo, in vivo and / or in vitro models; in some embodiments, such assessment or empirical determination is made in vivo, in a patient population (e.g., so that a correlation is established), or alternatively in a particular individual of interest.

[0122] In some embodiments, the IL-1 1 polypeptide and the second agent are administered according to an intermittent dosing regimen including at least two cycles. Where two or more agents are administered in combination, and each by such an intermittent, cycling, regimen, individual doses of different agents may be interdigitated with one another. In certain aspects, one or more doses of a second agent is administered a period of time after a dose of the first agent. In some embodiments, each dose of the second agent is administered a period of time after a dose of the first agent. In certain aspects, each dose of the first agent is followed after a period of time by a dose of the second agent. In some embodiments, two or more doses of the first agent are administered between at least one pair of doses of the second agent; in certain aspects, two or more doses of the second agent are administered between at least one pair of doses of the first agent. In some embodiments, different doses of the same agent are separated by a common interval of time; in some embodiments, the interval of time between different doses of the same agent varies. In certain aspects, different doses of the IL-1 1 polypeptide and the second agent are separated from one another by a common interval of time; in some embodiments, different doses of the different agents are separated from one another by different intervals of time.

[0123] One exemplary protocol for interdigitating two intermittent, cycled dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount the IL-1 1 polypeptide is administered to the individual; (b) a first resting period; (c) a second dosing period during which a therapeutically effective amount of the second agent is administered to the individual; and (d) a second resting period. A second exemplary protocol for interdigitating two intermittent, cycled dosing regimens may include: (a) a first dosing period during which a therapeutically effective amount the second agent is administered to the individual; (b) a first resting period; (c) a second dosing period during which a therapeutically effective amount of the IL-11 polypeptide is administered to the individual; and (d) a second resting period.

[0124] In some embodiments, the first resting period and second resting period may correspond to an identical number of hours or days. Alternatively, in some embodiments, the first resting period and second resting period are different, with either the first resting period being longer than the second one or, vice versa. In some embodiments, each of the resting periods corresponds to 120 hours, 96 hours, 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 30 hours, 1 hour, or less. In some embodiments, if the second resting period is longer than the first resting period, it can be defined as a number of days or weeks rather than hours (for instance 1 day, 3 days, 5 days, 1 week, 2, weeks, 4 weeks or more).

[0125] If the first resting period’s length is determined by existence or development of a particular biological or therapeutic event, then the second resting period’s length may be determined on the basis of different factors, separately or in combination. Exemplary such factors may include type and / or stage of a cancer against which the therapy is administered; properties (e.g., pharmacokinetic properties) of the IL-11 polypeptide, and / or one or more features of the patient’s response to therapy with the IL-1 1 polypeptide. In some embodiments, length of one or both resting periods may be adjusted in light of pharmacokinetic properties (e.g., as assessed via plasma concentration levels) of one or the other of the administered agents. For example, a relevant resting period might be deemed to be completed when plasma concentration of the relevant agent is below a pre-determined level, optionally upon evaluation or other consideration of one or more features of the individual’s response.

[0126] In certain aspects, the number of cycles for which a particular agent is administered may be determined empirically. Also, in some embodiments, the precise regimen followed (e.g., number of doses, spacing of doses (e.g., relative to each other or to another event such as administration of another therapy), amount of doses, etc.) may be different for one or more cycles as compared with one or more other cycles.

[0127] The IL-1 1 polypeptide and the second agent may be administered together or independently via any suitable route of administration. The IL-11 polypeptide and the second agent may be administered via a route of administration independently selected from oral, parenteral (e.g., by intravenous, intra-arterial, subcutaneous, intramuscular, or epidural injection), topical, or intra-nasal administration. According to certain embodiments, the IL-1 1 polypeptide and the second agent are both administered orally (e.g., in tablet form, capsule form, liquid form, or the like) either concurrently (in the same pharmaceutical composition or separate pharmaceutical compositions) or sequentially.

[0128] KITS

[0129] Aspects of the present disclosure further include kits. In certain embodiments, the kits find use in practicing the methods of the present disclosure, e.g., methods of treating a condition associated with IL-1 1 R signaling in a subject in need thereof.

[0130] Accordingly, in certain embodiments, a kit of the present disclosure comprises any of the IL-11 polypeptides of the present disclosure (e.g., present in a pharmaceutical composition), and instructions for administering the IL-1 1 polypeptide to an individual in need thereof. As will be appreciated, the kits of the present disclosure may include any of the IL-1 1 polypeptides having any of the features (e.g., targeting moieties, etc.) described above in the section relating to the IL-11 polypeptides of the present disclosure, which are not reiterated herein for purposes of brevity.

[0131] The kits of the present disclosure may include a quantity of the IL-1 1 polypeptide, present in unit dosages, e.g., ampoules, or a multi-dosage format. As such, in certain embodiments, the kits may include one or more (e.g., two or more) unit dosages (e.g., ampoules) of an IL-1 1 polypeptide of the present disclosure. The term “unit dosage”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the IL-1 1 polypeptide calculated in an amount sufficient to produce the desired effect. The amount of the unit dosage depends on various factors, such as the IL-1 1 polypeptide employed, the effect to be achieved, and the pharmacodynamics associated with the IL-11 polypeptide, in the individual. In yet other embodiments, the kits may include a single multi dosage amount of the IL-1 1 polypeptide.

[0132] The instructions (e.g., instructions for use (IFU)) included in the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet) are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0133] For purposes of completeness, non-limiting aspects and embodiments of the present disclosure are further disclosed in the following numbered clauses.

[0134] 1 . An interleukin 11 (IL-11) polypeptide, wherein the IL-11 polypeptide comprises: one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 receptor (IL-11 R) as compared to wild-type human IL-11 ; and one or more amino acid substitutions which inhibit formation of a complex comprising the IL-11 polypeptide, human IL-11 R and glycoprotein 130 (gp130) upon binding of the IL-11 polypeptide to human IL-11 R.

[0135] 2. The IL-11 polypeptide of clause 1 , wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 comprise a substitution at D19, T21 , L57, G62, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

[0136] 3. The IL-11 polypeptide of clause 2, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 R comprise D G, T211 , L57P, G62R, or any combination thereof.

[0137] 4. The IL-11 polypeptide of clause 2 or clause 3, comprising one, two, three or each of the D19, T21 , L57, and G62 substitutions.

[0138] 5. The IL-11 polypeptide of any one of clauses 1 to 4, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 R comprise a substitution at G71 , V72, or both.

[0139] 6. The IL-11 polypeptide of clause 5, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 comprise G71 D, V72M, or both.

[0140] 7. The IL-11 polypeptide of any one of clauses 1 to 6, wherein the one or more amino acid substitutions which inhibit formation of a complex comprising the IL-11 polypeptide, human IL-

[0141] 11 R and gp130 upon binding of the IL-11 polypeptide to human IL-11 R comprise a substitution at L24, W147, or both, wherein numbering of positions is according to SEQ ID NO: 1. 8. The IL-11 polypeptide of clause 7, wherein the one or more amino acid substitutions which inhibit formation of a complex comprising the IL-11 polypeptide, human IL-11 R and gp130 upon binding of the IL-11 polypeptide to human IL-11 R comprise L24R, W147A, or both.

[0142] 9. The IL-11 polypeptide of clause 8, comprising both of the L24 and W147 substitutions.

[0143] 10. The IL-11 polypeptide of any one of clauses 1 to 9, further comprising one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for mouse IL-11 R as compared to wild-type human IL-11 .

[0144] 11 . The IL-11 polypeptide of clause 10, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for mouse IL-11 R comprise a substitution at D41 , S60, D79, H86, G158, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

[0145] 12. The IL-11 polypeptide of clause 11 , wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for mouse IL-11 R comprise D41 E, S60T, D79Y, H86Y, G158E, or any combination thereof.

[0146] 13. The IL-11 polypeptide of clause 10, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for mouse IL-11 R comprise S60T.

[0147] 14. A nucleic acid encoding the IL-11 polypeptide of any one of clauses 1 to 13.

[0148] 15. An expression vector comprising the nucleic acid of clause 14 operably linked to a promoter.

[0149] 16. A host cell comprising the nucleic acid of clause 14 or the expression vector of clause 15.

[0150] 17. The host cell of clause 16, comprising the expression vector of clause 15.

[0151] 18. A method of producing the IL-11 polypeptide of any one of clauses 1 to 13, the method comprising culturing the host cell of clause 17 under conditions suitable for the cell to express the IL-11 polypeptide, wherein the IL-11 polypeptide is produced.

[0152] 19. A composition comprising the IL-11 polypeptide of any one of clauses 1 to 13.

[0153] 20. The composition of clause 19, comprising the IL-11 polypeptide formulated for administration to a subject.

[0154] 21 . The composition of clause 20, wherein the composition is formulated for parenteral administration to a subject.

[0155] 22. A method of treating a condition associated with IL-11 R signaling in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of clause 20 or 21 .

[0156] 23. The method according to clause 22, wherein the administering is by parenteral administration to the subject. 24. The method according to clause 22 or 23, wherein the condition associated with IL-11 R signaling is cancer.

[0157] 25. The method according to clause 24, wherein the cancer is a carcinoma, lymphoma, blastoma, or sarcoma.

[0158] 26. The method according to clause 24 or 25, wherein the cancer is a lung cancer, gastric cancer, colorectal cancer, breast cancer, or bone cancer.

[0159] 27. The method according to clause 26, wherein the cancer is lung adenocarcinoma.

[0160] 28. The method according to any one of clauses 24 to 27, wherein the condition is associated with IL-1 1 R signaling by cancer-associated fibroblasts (CAFs).

[0161] 29. The method according to any one of clauses 24 to 28, wherein the composition is administered to the subject in combination with chemotherapy.

[0162] 30. The method according to clause 29, wherein the composition is administered to the subject prior to, during and / or subsequent to administration of the chemotherapy.

[0163] 31 . The method according to clause 22 or 23, wherein the condition associated with IL-11 R signaling is fibrosis.

[0164] 32. The method according to clause 31 , wherein the fibrosis is fibrosis of the lung, liver, heart, or any combination thereof.

[0165] The following examples are offered by way of illustration and not by way of limitation.

[0166] EXPERIMENTAL

[0167] Introduction

[0168] Described herein is the use of the IL-1 1 ligand, which lacks binding to other IL-6 family member receptors,31as a starting point for developing a high affinity IL-11 R antagonist. Combinatorial methods enabled development of an engineered IL-11 cytokine with ~70-fold higher affinity for IL-11 R as compared to wild-type IL-1 1 . Introduction of two additional point mutations ablated binding to the gp130 co-receptor at two putative IL-1 1 binding sites, resulting in an antagonist that disrupts the active signaling complex and decreases lung adenocarcinoma progression in a mouse tumor model.

[0169] Example 1 - Engineering agonist IL-11 variants with increased affinity for IL-1 1 receptor

[0170] Combinatorial screening via yeast surface display was used to first identify human IL-1 1 variants with improved affinity for the human IL-1 1 receptor (IL-1 1 R) extracellular domain. IL-1 1 was displayed on the surface of yeast cells through genetic fusion to the agglutinin mating protein (Aga2p), which complexes with Aga1 p contained in the yeast strain EBY100.3233Yeast-displayed IL-11 expression was detected via flow cytometry using antibodies specific to a C-terminal c-myc tag, and IL-1 1 R binding was detected via antibodies against a 6x histidine tag (Figure 1a). A mutagenic library was created by error-prone PCR of the gene coding for human IL-11 ,34and the resulting DNA was electroporated into yeast strain EBY100 by homologous recombination to yield a library of ~5x107transformants.35Three successive rounds of flow cytometric sorting were performed on yeast displaying individual IL-1 1 variants, collecting clones with the strongest binding to soluble human IL-11 R during each sort (Figure 1 b). Pools of isolated yeast clones were propagated in culture following each sort round, and IL-1 1 genes contained in individual yeast from the final sort (post-Sort 1.3) were sequenced. This analysis identified a consensus mutation (L57P) with an apparent binding affinity (Kd) of 0.71 + / - 0.17 nM, a ~10x improvement in human IL-11 R binding compared to wild-type human IL-11 (Figure 6a-b).

[0171] A second-generation IL-1 1 library was created by DNA shuffling, based on the pool of variants isolated from the first library screen (post-Sort 1 .3), using staggered extension process (StEP) to recombine the mutations.36In parallel, new mutations were introduced via error-prone PCR. The resulting yeast-displayed library of ~107yeast transformants was screened using four rounds of flow cytometric sorting of increasing stringency, again collecting the strongest human IL-11 R binding clones (Figure 7a). The final isolated library pool (post-Sort 2.4) bound to IL-1 1 R more tightly than either of the initial libraries and the post-Sort 1.3 pool (Figure 1c). DNA sequencing of post-Sort 2.3 and 2.4 revealed six consensus mutations (D19G, T211, L57P, G62R, G71 D, and V72M) that appeared in multiple clones (Figure 1d). Yeast-displayed IL-11 mutants were individually tested for binding to soluble IL-11 R in an additive manner built upon L57P (‘P’) as ‘IP’, ‘IPR’, ‘GIPR’, ‘GIPRD’, and ‘GIPRDM’ (Figure 2a, Figure 7b). We found that the IL-11 GIPR variant had the tightest apparent Kdof 0.12 + / - 0.01 nM, a ~70x improvement over wild-type IL-11 (Figure 2b). The L57P and G62R mutations fall within a region of IL-1 1 (termed Site I) previously described to confer binding to IL-1 1 R (Figure 2c).37To investigate the effect of IL-1 1 affinity on downstream signaling, a HeLa reporter cell line containing the firefly luciferase gene under control of a pSTAT3 inducible response element was transduced with full- length IL-1 1 R. We show that the IL-11 GIPR variant is an agonist cytokine that induces increased pSTAT3 signaling as compared to wild-type IL-11 in the HeLa reporter cells and A549 human non-small cell lung cancer (NSCLC) (Figure 2d-e). We hypothesize that the increased maximal HeLa pSTAT3 signal induced by IL-1 1 GIPR is due to increased receptor occupancy and residence time driven by the higher affinity compared to wild-type IL-1 1 .

[0172] Example 2 - Engineering high-affinity IL-11 variants with loss of binding to gp130 co-receptor

[0173] Next, to develop an IL-11 R antagonist, we proceeded to create an IL-11 cytokine that retains high affinity receptor binding but abrogates gp130 complex formation. IL-1 1 is proposed to signal through a hexameric complex of IL-1 1 R and gp130. This complex forms through an initial binding event between IL-1 1 and IL-11 R that predisposes the complex to bind with high affinity to gp130 and promote downstream signaling.10Previously, a single point mutation, W147A, was reported to reduce binding of IL-11 to gp130, converting IL-11 from an agonist to an antagonist.38Here, we found that IL-1 1 GIPR W147A exhibited reduced, but still significant binding to gp130 when the variant was displayed on the surface of yeast and pre-complexed with soluble IL-11 R (Figure 3a, d). Thus, we generated and screened an additional IL-1 1 yeast library (~108transformants) to identify a more complete gp130 binding-deficient mutant as an effective receptor antagonist. In this library, error-prone PCR was used to introduce a low frequency of mutations to DNA templates encoding for IL-11 GIPR and GIPR W147A in a 50 / 50 mixture. Two rounds of ‘negative’ screens were performed by flow cytometric sorting, where the yeast- displayed IL-1 1 library was pre-complexed with a saturating concentration of IL-11 R before adding soluble gp130. The population of yeast clones that expressed IL-1 1 but did not bind gp130 were collected in each of these sorts, followed by a ‘positive’ screen for IL-1 1 R binding-competent clones to ensure that the overall IL-1 1 structural integrity was not disrupted by mutations. This alternating ‘negative-positive’ screening strategy was repeated with increasing concentrations of gp130 and a reduced concentration of IL-1 1 R (Figure 3b). After six rounds of sorting, DNA sequencing of yeast clones identified two consensus mutations: L24R and the initial W147A mutation (Figure 3c). In further analysis, we showed that IL-11 variants containing GIPR and GIPR W147A maintain significant gp130 binding whereas GIPR L24R W147A has minimal binding to gp130 (Figure 3d). Mapping the mutations onto the crystal structure of IL-1 1 (PDB: 6040),31we observed that L24R is located in a region known as ‘Site II’, one of two proposed gp130 binding sites on IL-1 1 , while W147A is located in the ‘Site III’ region, the other proposed gp130 binding site (Figure 3e,f).10’39Thus, it is intriguing to postulate that the combination of the L24R and W147A mutations across two purported binding sites allows for more complete reduction of gp130 binding.

[0174] Example 3 - Engineering a human / mouse cross-reactive IL-11 variant

[0175] Wild-type human IL-11 and mouse IL-1 1 (mlL-11 ) bind to mouse IL-1 1 R (mlL-1 1 R) with similar low nanomolar affinity (Figure 4a). Our engineered IL-1 1 GIPR variant did not show increased binding affinity to mlL-1 1 R (Figure 4a), indicating that affinity maturation against human IL-11 R did not consequentially confer tighter binding to mlL-1 1 R. Importantly, however, the L24R and W147A mutations also ablated mouse gp130 binding (Figure 4b), demonstrating that IL-1 1 GIPR L24R W147A is effective in preventing formation of the mouse IL-1 1 / IL- 1 1 R / gp130 hexameric complex.

[0176] To evaluate therapeutic efficacy more effectively in mouse lung cancer models, we sought to engineer an IL-1 1 antagonist that has higher affinity for hlL-1 1 R and mlL-1 1 R. Using the yeast- displayed GIPR library described above, we performed five rounds of flow cytometric sorting against decreasing concentrations of mlL-1 1 R (Figure 8a). Five consensus mutations were identified based on DNA sequencing of the post-Sort 4 and 5 yeast pools (D41 E, S60T, D79Y, H86Y, and G158E) (Figure 8b). Desiring introduction of a minimally perturbing mutation, we selected to advance S60T, which like the L57P and G62R mutations lies in proximity to the proposed Site I IL-1 1 R binding region. When added to IL-11 GIPR, S60T was shown to bind to mlL-11 R with an apparent Kd Of 0.29 + / - 0.01 nM, an ~10x improvement over wild-type mlL-1 1 binding (Figure 4c).

[0177] Collectively, our final engineered variant of human IL-1 1 (termed enlL-1 1 ) has four mutations to improve the affinity to hlL-1 1 R (D G, T211, L57P, and G62R); one mutation to improve the affinity to mlL-11 R (S60T); and two mutations to ablate gp130 binding (L24R and W147A) (Figure 4d). The binding affinity of en IL-1 1 for IL-1 1 R and mlL-11 R was measured via biolayer interferometry (BLI) to be 410 + / - 10 pM and 160 + / - 10 pM respectively (Figure 9a, b).

[0178] Example 4 - enlL-11 inhibits cell signaling and slows tumor growth

[0179] To directly parse the effects of improved receptor binding affinity, enlL-1 1 was compared to a variant of wild-type human IL-11 containing gp130-disrupting mutations L24R and W147A (termed IL-1 1 RA). The HeLa reporter cell line described above was used to interrogate the ability of the IL-1 1 variants to compete with wild-type IL-1 1 to block downstream pSTAT3 signaling. enlL-1 1 and IL-1 1 RA were added to the reporter cells at varying concentrations simultaneously with 0.5 nM of WT IL-11 . enlL-1 1 more completely ablated pSTAT3 signaling with an IC50 of 0.7 + / - 0.1 nM as compared to IL-1 1 RA, which showed minimal signal inhibition (Figure 5a). Next, A549 cells were incubated with 20 nM of wild-type IL-1 1 and increasing concentrations of IL-11 RA and enlL-1 1 . Notably, enlL-11 significantly decreased pSTAT3 levels at 1 nM and 10 nM of inhibitor compared to IL-1 1 RA and was able to reduce pSTAT3 levels to baseline (Figure 5b). This reduction in signaling was also seen for phospho-ERK under the same conditions (Figure 10a).

[0180] The therapeutic efficacy of enlL-11 was next tested in a mouse xenograft model of NSCLC using A549 cells engrafted into the flank of immunodeficient nude mice. Tumors were allowed to establish to an average volume of 100 mm3before mice were binned into groups for treatment (Figure 11a). Mice were injected intraperitoneally with 1 mg / kg or 5 mg / kg of enlL-1 1 or a saline control three times a week for four weeks. No significant change in mouse body weight was observed between the treated and untreated groups (Figure 11 b). Mice treated with 5 mg / kg saw significantly slower tumor growth compared to saline treated mice (Figure 5c). This reduction in tumor growth was observed in a dose-dependent manner (Figure 11 c-d). Tumors were excised and stained for the proliferation marker, phospho-histone H3 (PH3) and found to have a significant reduction in positive nuclei in the treated group (Figure 5d). Collectively, these results show that an engineered IL-1 1 cytokine with mutations that increase IL-1 1 R binding affinity and ablate gp130 binding can function as a potent decoy ligand in cell culture and animal models. Materials and Methods

[0181] Preparation of IL-11 libraries. DNA encoding IL-1 1 (residues Pro22 to Leu199) was cloned into the yeast display vector pCTcon2 (41843, Addgene) using Nhelanti BamHI restriction sites. This was used as the template for error-prone PCR using Taq polymerase (50-81 1 -694, Fisher Scientific), 50 mM MgCh, and varying concentrations (1 .25, 2.5, 5, 10 pM) of dNTP analogs 2’-dPTP (N-2037, TriLink Biotechnologies) and 8-oxo-2’-dGTP (N-2034, TriLink Biotechnologies). The PCR products were pooled, amplified using Phusion polymerase, and purified by gel electrophoresis. The cDNA and linearized plasmid were electroporated into EBY100 yeast where they were assembled via homologous recombination. Library size was estimated to be 5x107via dilution plating. The second generation library was generated using the post-Sort 1 .3 pool as the template for error-prone PCR and DNA shuffling using StEP36. The extension protocol was run for 80 cycles with 94 °C for 30 s and 55 °C for 5 s. The error-prone and StEP products were purified by gel electrophoresis and electroporated with linearized plasmid into EBY100 yeast. Library size was estimated to be 107by dilution plating. The GIPR / GIPR W147A library was created using Mutazyme II DNA polymerase (200550, Agilent) with 1 pg of DNA as template. The library size was estimated to be 108.

[0182] Library screening. IL-11R screening. Yeast displaying high-affinity IL-1 1 variants were isolated using FACS on a Sony sorter. The seven total rounds of screening against IL-1 1 R (Sino Biological, 10252-H08H) were performed using equilibrium binding sorts by incubating the yeast library at room temperature in phosphate-buffered saline (PBS) with 1 mg / mL bovine serum albumin (BSA) (BPBS) with the following concentrations of IL-11 R: 50 nM for Sort 1.1 , 1 nM for Sort 1 .2, 250 pM for Sort 1 .3, 5nM for Sort 2.1 , 500 pM for Sort 2.2, 100 pM for Sort 2.3, and 50 pM for Sort 2.4. After the incubation with IL-11 R, yeast were washed with BPBS, pelleted, and resuspended in BPBS with a 1 :5000 dilution of chicken anti-c-myc (A21281 , Invitrogen) for 30 min at 4 °C. Yeast were washed, pelleted, and resuspended with 1 :500 dilutions of goat antichicken AF488 (A11039, Fisher Scientific) and mouse anti-his AF647 (MA1 135A647, Thermo Scientific) for 15 min at 4 °C. The labeled yeast were sorted by FACS on a Sony SH800 Sorter with a minimum of 10x diversity coverage and the strongest binding clones based on the ratio of IL-11 R binding to c-myc expression were collected. The sorted clones were propagated, induced, and used for the next round of screening. Plasmid DNA was recovered using a Zymoprep kit (D2001 , Zymo Research Corp), transformed into DH10B electrocompetent cells, and isolated using a plasmid miniprep kit (K0503, Thermo Fisher). Sequencing was performed by MCLAB. gp130 / IL-11R screening. Yeast displaying the GIPR / GIPR W147A library were treated as described in the previous section with the following conditions: 100 nM IL-11 R and 5 nM gp130- Fc (Sino Biological, 10974-H03H-20) for Sort 3.1 ; 100 nM IL-1 1 R and 10 nM gp130-Fc for Sort 3.2; 1 nM IL-1 1 R for Sort 3.3; 100 nM IL-11 R and 20 nM gp130-Fc for Sort 3.4; 100 nM IL-11 R and 40 nM gp130-Fc for Sort 3.5; 250pM IL-11 R for Sort 3.6. Incubations with IL-1 1 R were performed overnight, washed, and then gp130-Fc was added for three hours before staining as described above with goat anti-human AF647 (SouthernBiotech, 2040-31 ). mlL-11R screening. Yeast displaying the GIPR / GIPR W147A library were treated as described in the previous section with the following conditions: 5 nM mlL-1 1 R (Sino Biological, 50075-M08H-100) for Sort 4.1 , 1 nM mlL-1 1 R for Sort 4.2; 250 pM mlL-1 1 R for Sort 4.3; 200 pM mlL-11 R for Sort 4.4; 150 pM mlL-11 R for Sort 4.5. Incubations with mlL-1 1 R were performed overnight, washed, and then stained as described above.

[0183] Yeast-surface display binding assays. Yeast-displayed IL-1 1 variants were incubated with varying concentrations of IL-11 R or mlL-11 R overnight at room temperature to reach equilibrium, washed with BPBS, pelleted, and stained with a 1 :5000 dilution of chicken anti-c- myc for 30 min at 4 °C. The yeast were washed with BPBS, pelleted, and stained with a 1 :500 dilution of goat anti-chicken AF488 and mouse anti-his AF647 for 15 min at 4 °C. The samples were washed and analyzed by flow on a Accuri C6 cytometer (BD Biosciences) and data was analyzed using FlowJo. To characterize binding to gp130, yeast-displayed IL-1 1 variants were incubated with 100 nM of IL-11 R or mlL-1 1 R for a minimum of 3 hours, washed with BPBS, pelleted, and resuspended with varying concentrations of gp130-Fc. The gp130-Fc was detected with a 1 :500 dilution of goat anti-human AF647.

[0184] Protein expression. cDNA of IL-1 1 with an N-terminal fusion of 6x-histidine tag, thioredoxin, and TEV protease cleavage site was cloned into the pET28 plasmid with an inducible lac promoter and amplified in DH10B cells. Purified plasmid was transformed into BL21 cells. Cells at an OD between 0.5-0.8 were induced with 0.1 mM isopropyl-B-o-thiogalactoside (Gold Biotechnology, 12481 C25) in LB overnight at 30 °C. Induced cells were pelleted and resuspended in a phosphate buffer with 10 mM imidazole (Sigma-Aldrich, I2399-500G). The cell resuspension was frozen at -80C for 20 minutes and then allowed to thaw at room temperature. The resuspension was sonicated for 10 minutes and then centrifuged for 20 minutes at 10000 rpm. The supernatant was purified using cobalt beads (H-310-50, Gold Biotechnology). Protein was concentrated and buffer-exchanged into PBS using Amicon Ultra-15 centrifugal filter units (UFC901024, Millipore-Sigma, 10 kDA cut-off). Protein was cleaved overnight at 4 °C with TEV protease (P81 12S, NEB) and purified with cobalt beads. The cleaved IL-1 1 was collected in the flowthrough and buffer exchanged into PBS. Concentration was measured using a NanoDrop 2000 (Thermo Fisher).

[0185] Biolayer interferometry. Biotinylated IL-11 R-Fc (ILR-H82F5, Aero Biosystems) or mlL- 1 1 R-mFc (7405-MR, R&D Systems) was loaded onto streptavidin coated biosensors (19-5019, ForteBio) or anti-mouse Fc biosensors (18-5088, ForteBio) at 1 .25 ug / mL in kinetics buffer (18- 1 105, Sartorius). Three-fold serial dilutions of WT IL-11 or enlL-11 were made in kinetics buffer. Baseline measurement was performed in kinetics buffer alone, then association in the WT IL-1 1 or en IL-1 1 conditions followed by dissociation in kinetics buffer alone. Data was collected with an Octet QK (ForteBio) and analyzed using the Data Analysis software. HeLa STAT3 luciferase reporter cell assays. A STAT3 luciferase reporter HeLa stable cell line (SL-0003-FP, Signosis) was transfected with full length IL-1 1 R and cultured in DMEM + 10% FBS + 100 pg / mL of Hygromycin B (Fisher Scientific, 10-687-010). Cells were seeded 16 hours before the assay into 96-well plates at 1 .5 x 104cells / well. The culture medium was switched to DMEM with 0.1 % FBS for 2 hours prior to adding the cytokines. Dilutions of the cytokines were made in DMEM with 0.1 % FBS. For agonism studies, varying concentrations of IL-11 or GIPR were added. For antagonism studies, 0.5 nM of IL-1 1 was added to all conditions simultaneously with varying concentrations of inhibitor. Cytokines were incubated on the cells at 37 °C for 4 hours. To quantify luciferase activity, cells were lysed with 20 pL of Lysis Buffer (E297A, Promega) for 10 min and then transferred to a white bottom 96-well plate. Luciferase assay substrate (E151 A, Promega) was resuspended in luciferase assay buffer (E152A, Promega) and 100 pL was added to each well. Luminescence was measured on a BioTek Synergy H4 plate reader.

[0186] Phospho-STAT3 and phospho-ERK signaling assays. A549 cells were seeded the day before the assay in a 12-well plate at 1 .5 x 105cells per well. Cells were starved in serum- free media for two hours. For phospho-ERK assays, 10 pM U0126 (9903S, Cell Signaling Technology) was added to reduce baseline levels. For agonism assays, IL-11 or GIPR was added at indicated concentrations. Cytokines were diluted in serum-free media with 0.1% BSA. For antagonism assays, IL-11 was added at 20 nM simultaneously with the indicated concentration of IL-1 1 RA or enlL-1 1. Cytokines were added to the serum-starved cells for 20 min at 37 °C. Cells were washed twice with cold PBS and then 100 pL of RIPA Lysis and Extraction buffer (89900, Fisher Scientific) with phosphatase inhibitor r (P5726, Sigma-Aldrich) and protease inhibitor (P8340, Sigma-Aldrich) was added to each well for 10 min. Protein equivalent amounts of protein (15-20 pg) were ran on a PAGE gel. Samples were transferred to a nitrocellulose membrane (IB301031 , Thermo Fisher) with an iBIot dry blotting system. Blots were blocked overnight in TBST with 5% BSA. STAT3 signaling blots were stained with 1 :4000 phospho-stat3 (pY705) rabbit antibody (9145S, Fisher Scientific) and 1 :5000 anti-p-tubulin mouse antibody (50- 103-0148, Fisher Scientific), or 1 :5000 Stat3 (D3Z2G) rabbit antibody (NC0969631 , Fisher Scientific) in TBST + 5% BSA for two hours at room temperature. ERK signaling blots were stained with 1 :5000 phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (9101 , Cell Signaling Technology) and 1 :5000 STAT3 antibody or 1 :5000 p44 / 42 MAPK (Erk1 / 2) (9102, Cell Signaling Technology). Blots were washed three times for 5 min each with TBST. Secondary staining was done with 1 :5000 HRP-conjugated anti-rabbit (71 1 -035-152, Jackson ImmunoResearch) and anti-mouse antibodies (715-035-150, Jackson ImmunoResearch) in TBST + 5% BSA for 1 hour at room temperature before being washed again. Blots were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (34095, Fisher Scientific) and chemiluminescence was detected using a ChemiDoc XRS System (Bio-Rad). Images were quantified using ImageJ. In vivo tumor model. A549 cells (106) mixed 1 :1 with Matrigel for a total volume of 100 pL (CB-40230, Fisher Scientific) were injected subcutaneously in the right flank of 8-week-old nude mice (NU / J 002919, The Jackson Laboratory). Tumors were allowed to grow to an average of 100mm3before being stratified into treatment groups. enlL-11 (GIPR RA S60T) in saline was injected intraperitoneally three times a week at 1 mg / kg and 5 mg / kg. Saline alone was used as the control. The tumor volume was measured by digital calipers three times per week and calculated using volume = (length) x (width) x 0.5(width). Tumors were excised at the end of the study and fixed in 10% buffered formalin for 24 hours and then stored in 70% ethanol before paraffin embedding. Immunohistochemistry staining was done by HistoTec Labs.

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[0250] 18;8(6):e10573. doi: 10.1002 / btm2.10573 - the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0251] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:

1. An interleukin 11 (IL-11) polypeptide, wherein the IL-11 polypeptide comprises: one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 receptor (IL-11 R) as compared to wild-type human IL-11 ; and one or more amino acid substitutions which inhibit formation of a complex comprising the IL-11 polypeptide, human IL-11 R and glycoprotein 130 (gp130) upon binding of the IL-11 polypeptide to human IL-11 R.

2. The IL-11 polypeptide of claim 1 , wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 R comprise a substitution at D19, T21 , L57, G62, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

3. The IL-11 polypeptide of claim 2, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 R comprise D19G, T21 I, L57P, G62R, or any combination thereof.

4. The IL-11 polypeptide of claim 2 or claim 3, comprising one, two, three or each of the D19, T21 , L57, and G62 substitutions.

5. The IL-11 polypeptide of any one of claims 1 to 4, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 R comprise a substitution at G71 , V72, or both.

6. The IL-11 polypeptide of claim 5, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for human IL-11 R comprise G71 D, V72M, or both.

7. The IL-11 polypeptide of any one of claims 1 to 6, wherein the one or more amino acid substitutions which inhibit formation of a complex comprising the IL-11 polypeptide, human IL- 11 R and gp130 upon binding of the IL-11 polypeptide to human IL-11 R comprise a substitution at L24, W147, or both, wherein numbering of positions is according to SEQ ID NO: 1.

8. The IL-11 polypeptide of claim 7, wherein the one or more amino acid substitutions which inhibit formation of a complex comprising the IL-11 polypeptide, human IL-11 R and gp130 upon binding of the IL-11 polypeptide to human IL-11 R comprise L24R, W147A, or both.

9. The IL-11 polypeptide of claim 8, comprising both of the L24 and W147 substitutions.

10. The IL-11 polypeptide of any one of claims 1 to 9, further comprising one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for mouse IL-11 R as compared to wild-type human IL-11 .11 . The IL-11 polypeptide of claim 10, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for mouse IL-11 R comprise a substitution at D41 , S60, D79, H86, G158, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

12. The IL-11 polypeptide of claim 11 , wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for mouse IL-11 R comprise D41 E, S60T, D79Y, H86Y, G158E, or any combination thereof.

13. The IL-11 polypeptide of claim 10, wherein the one or more amino acid substitutions which increase affinity of the IL-11 polypeptide for mouse IL-11 R comprise S60T.

14. A nucleic acid encoding the IL-11 polypeptide of any one of claims 1 to 13.

15. An expression vector comprising the nucleic acid of claim 14 operably linked to a promoter.

16. A host cell comprising the nucleic acid of claim 14 or the expression vector of claim 15.

17. The host cell of claim 16, comprising the expression vector of claim 15.

18. A method of producing the IL-11 polypeptide of any one of claims 1 to 13, the method comprising culturing the host cell of claim 17 under conditions suitable for the cell to express the IL-11 polypeptide, wherein the IL-11 polypeptide is produced.

19. A composition comprising the IL-11 polypeptide of any one of claims 1 to 13.

20. The composition of claim 19, comprising the IL-11 polypeptide formulated for administration to a subject.21 . The composition of claim 20, wherein the composition is formulated for parenteral administration to a subject.

22. A method of treating a condition associated with IL-11 R signaling in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 20 or 21 .

23. The method according to claim 22, wherein the administering is by parenteral administration to the subject.

24. The method according to claim 22 or 23, wherein the condition associated with IL-11 R signaling is cancer.

25. The method according to claim 24, wherein the cancer is a carcinoma, lymphoma, blastoma, or sarcoma.

26. The method according to claim 24 or 25, wherein the cancer is a lung cancer, gastric cancer, colorectal cancer, breast cancer, or bone cancer.

27. The method according to claim 26, wherein the cancer is lung adenocarcinoma.

28. The method according to any one of claims 24 to 27, wherein the condition is associated with IL-11 R signaling by cancer-associated fibroblasts (CAFs).

29. The method according to any one of claims 24 to 28, wherein the composition is administered to the subject in combination with chemotherapy.

30. The method according to claim 29, wherein the composition is administered to the subject prior to, during and / or subsequent to administration of the chemotherapy.31 . The method according to claim 22 or 23, wherein the condition associated with IL-11 R signaling is fibrosis.

32. The method according to claim 31 , wherein the fibrosis is fibrosis of the lung, liver, heart, or any combination thereof.

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