NZ748787B2 - Compositions and methods for modulating il-10 immunostimulatory and anti-inflammatory properties - Google Patents

Abstract

The invention provides compositions and methods for modulating the immunostimulatory properties and/or anti-inflammatory properties of IL-10. The present invention provides scIL-10 polypeptides of Formula 1. The polypeptides of the invention are optionally linked to a fusion partner.

Description

COMPOSITIONS AND METHODS FOR MODULATING IL-lO IMMUNOSTIMULATORY AND ANTI-INFLAMMATORY PROPERTIES OUND OF THE INVENTION IL-10 is considered a potent anti-inflammatory cytokine that strongly ts the production of inflammatory mediators. However, recent studies have suggested that IL-10 also has immunostimulatory properties on CD4+, CD8+ T cells, and/or NK cells, resulting in increased IFN-y production which in turn may lead to related inflammatory responses in humans.

Despite encouraging pre-clinical data ting this cytokine as therapeutically le biological, results of clinical trials evaluating the merit of IL-10 administration in c inflammation have been preponderantly ointing. Bulk of pre-clinical data and analysis of patients with IL-10 or IL-10 or defects clearly point to endogenously produced IL-lO as potent and significant anti-inflammatory determinant. However, thorough analysis further suggests that IL-10 has the potential to acquire sharply contrasting properties in an atory environment in vivo. In recent years several studies have been performed in order to verify the human response upon IL-lO administration, particularly in view of its anti-inflammatory potential. Those clinically important studies disclosed xing pro- inflammatory functions of IL-10. However, the basis of IL-10 immunostimulatory action s unclear.

On the other hand IL-lO has been explored for use in the treatment of proliferative disorders, e.g., cancer, tumors, etc. IL-lO induces cytotoxic activity of CD8 T-cells, antibody production of B-cell and suppresses macrophage activity and tumor promoting ation.

IL-lO appears to increase the infiltration of CD8+ T cells to a tumor, as well as increasing the expression of inflammatory nes that play a role in tumor immunity. Treatment with IL- may provide a significant improvement for tumor treatment.

One drawback of using IL-10 and particularly any form of recombinant IL-10 in therapy is its short serum half-life. One strategy for increasing serum half-life of a therapeutic protein such as IL-10 is to attach the protein to an Fc (fragment crystallizable) domain of an antibody. Many such fusion proteins are capable of forming homodimers or dimers thereby forming antibody-like fusion protein molecules.

ORATED BY REFERENCE (RULE 20.6) WO 05226 Depending on the therapeutic application, the ability to selectively enhance either the anti-inflammatory activity or the immunostimulatory activity of IL-10 would be desired. It would also be desirable to increase the half-life of recombinant IL-10.

SUMMARY OF THE INVENTION The invention provides compositions and methods for modulating the immunostimulatory properties and/or anti-inflammatory properties of IL-10. The present invention provides scIL—lO polypeptides of Formula 1. The polypeptides of the invention are optionally linked to a fusion partner.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects, features and ages of the invention will be apparent from the following more particular ption of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale emphasis instead being placed upon illustrating the principles of the invention. is a diagram of an Fc fusion protein homodimer of two polypeptide chains, n in each polypeptide chain comprises as X, scIL-lO which is then fused to the Fc region of an IgG1 dy via an scCLCHl linker. is a diagram of an F0 fusion protein homodimer of two polypeptide , wherein in each polypeptide chain comprises scIL-lO which is then fused to the Fc region of an IgG1 antibody via the novel scCHlCL . is an SDS-PAGE showing expression of an Fc fusion protein comprising scIL- fused to the Fc region of an IgG1 antibody via the novel scCLCHl linker (left) or via the novel scCHlCL linker (right) under reducing and non-reducing conditions. is a chromatogram showing the characterization of the IL-10 fused to the Fc region of an IgG1 antibody via the novel l linker by analytical gel filtration. is a chromatogram showing the characterization of the IL-lO fused to the Fc region of an IgG1 antibody via the novel scCHlCL linker by ical gel filtration. is a graph showing stimulation of mouse mast cell line MC/9 by the IL-10 single chain fusion proteins of the invention as compared to the scIL-lO direct Fc fusion protein used as a l. is a tic of the effects of amino acid tutions that disrupt either one or both of the two IL-10R1 interfaces (SEQ ID NOS: 20, 21 and 22).

INCORPORATED BY REFERENCE (RULE 20.6) is a schematic of the effects of amino acid substitutions that disrupt either one or both of the two IL-10R2 interfaces (SEQ ID NOS: 23, 24 and 25). is a schematic of the effects of amino acid substitutions that simultaneously disrupt one of the IL-lORl and one of the IL-10R2 interfaces. (SEQ ID NOS: 26-29).

DETAILED DESCRIPTION OF THE INVENTION Definitions By “polypeptide” is meant any sequence of two or more amino acids, regardless of length, post-translation modification, or function. “Polypeptide, 7) (4peptide,” and “protein” are used interchangeably herein. Polypeptides can include l amino acids and non-natural amino acids. Polypeptides can also be modified in any of a variety of standard chemical ways (e.g., an amino acid can be modified with a protecting group, the carboxy-terminal amino acid can be made into a terminal amide group; the amino-terminal residue can be modified with groups to, e. g, enhance ilicity, or the ptide can be chemically I5 glycosylated or otherwise modified to increase stability or in vivo half-life). Polypeptide modifications can include the attachment of r structure such as a cyclic compound or other molecule to the polypeptide and can also include polypeptides that contain one or more amino acids in an d configuration (i.e., R or S, or, L or D).

As used herein, “antibody” and “immunoglobulin” are used interchangeably and refer to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an antigen. Identified immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, n and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Terms understood by those in the art of antibody logy are each given the meaning acquired in the art, unless expressly defined differently herein. dies are known to have variable s, a hinge region, and constant domains. globulin structure and function are reviewed, for example, in Harlow et al, Eds., Antibodies: A Laboratory Manual, r 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

INCORPORATED BY REFERENCE (RULE 20.6) “Percent (%) amino acid sequence identity” herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the ces and introducing gaps, if necessary, to e the maximum t sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence ty can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN—Z or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring ent, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

The notations “mg/kg”, or “mg per kg” refer to milligrams per kilogram. All notations are used interchangeably throughout the present disclosure.

The “half-life” of a polypeptide can generally be defined as the time taken for the serum concentration of the ptide to be d by 50%, in viva, for example due to degradation of the polypeptide and/or clearance or sequestration of the polypeptide by natural mechanisms. The half-life can be determined in any manner known per se, such as by cokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may, for example, generally involve the steps of stering a suitable dose of a polypeptide to a rodent or primate, collecting blood samples or other samples from a rodent or primate at regular intervals; determining the level or concentration of the polypeptide in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the polypeptide has been reduced by 50% compared to the initial level upon dosing. Methods for determining half-life may be found, for example, in Kenneth et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists (1986), Peters et al, Pharmacokinete analysis: A Practical ch (1996); and “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).

The half-life of a fusion ptide is increased if presence in a biological matrix (blood, serum, plasma, tissue) persists, in viva, for a longer period as ed to an appropriate l. Half-life may be increased by 10%, 20%, 30%, 40%, 50% or more as compared to an appropriate l.

Half-life can be expressed using parameters such as the t1/2—a1pha, eta, and HL_Lambda_z. In the present specification, an “increase in half-life” refers to an se in any one of these parameters, any two of these parameters, or all three of these parameters.

An “increase in half-life” in particular refers to an increase in the ti/2-beta and]or INCORPORATED BY REFERENCE (RULE 20.6) HL_Lambda_z, either with or without an se in the ti/2-alpha. Other PK parameters that can be assessed include volume of distribution (VD), clearance (CL), and mean residence time (MRT), and the area under the curve (AUC). In the present specification, a “change in pharmacokinetics” refers to changes in any one of these parameters, any two of these parameters, any three of these parameters, or all four of these parameters, in the ce or absence of changes in the half-life parameters listed above.

“Activity” for the purposes herein refers to an action or effect of a component of a fusion protein consistent with, but not necessarily identical to, that of the corresponding native active protein, wherein “biological activity” or “bioactivity” refers to an In vitro or in viva biological function or effect, including but not limited to receptor binding, antagonist activity, t activity, or a cellular or physiologic response.

As used herein, a "dimer complex" comprises two single chains of sc-IL-10, or sc-IL- fused to an appropriate fusion r such as, for example, the scIL-lO-Ll-HINGE—Fc fusion proteins of the invention, wherein the two single chain polypeptides are associated together under appropriate conditions via either non-covalent binding or covalent g, for example, by a disulfide bridge. A "heterodimeric protein", “heterodimerized x”, or “heterodimer” as used interchangeably herein refers to a protein that is made of two single chain scIL-lO-Ll-HINGE-Fc polypeptides g a dimer x, wherein said two single chain ptides have different amino acid sequences. For example, one single chain peptide of the heterodimer has an scIL-lO based on Formula lwith at least one amino acid substitution and the other single chain peptide of the dimer has an scIL-10 sequence based on Formula 1 with no amino acid substitutions. A "homodimeric protein" “homodimerized complex” or “homodimer” as used interchangeably herein, refers to a protein that is made of two identical or ntially identical polypeptides forming the dimer complex, wherein said two single chain polypeptides preferably share 100% ty. There are circumstances, especially with regard to larger polypeptides wherein a homodimer comprises two ntially identical polypeptides having at least about 95% or at least about 99% identity, wherein any amino acid differences between the two polypeptide chains comprise amino acid substitutions, additions or deletions which do not affect the functional and physical properties of the polypeptide compared to its partner polypeptide of the homodimer such as, for example, conservative amino acid substitutions.

As used , a protein is "soluble" when it lacks any transmembrane domain or protein domain that anchors or integrates the polypeptide into the membrane of a cell expressing such polypeptide.

INCORPORATED BY REFERENCE (RULE 20.6) As used herein, “Fc domain”, “Fc region” or “Fc portion” as those terms may be used interchangeably herein to describe an scIL-lO-Ll-HINGE-Fc fusion protein of the invention, encompasses domains derived from the constant region of an immunoglobulin, preferably a human immunoglobulin, including a nt, analog, variant, mutant or derivative of the constant region. Suitable immunoglobulins include IgGl, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE and IgM. The constant region of an immunoglobulin is defined as a naturally-occurring or synthetically-produced polypeptide gous to the immunoglobulin C-terminal region, and can include a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately or in combination.

As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” is used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant ation or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an ement is ed in the subject, hstanding that the subject may still be afflicted with the underlying disorder.

For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

A peutic effect”, as used herein, refers to a physiologic , including but not limited to the cure, mitigation, amelioration, or prevention of disease in humans or other animals, or to ise e physical or mental well-being of humans or animals, caused by a fusion protein of the invention.

The terms “therapeutically effective amount” and “therapeutically ive dose”, as used herein, refers to an amount of an active protein, either alone or as a part of a fusion protein composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial.

The term “therapeutically ive dose n”, as used herein, refers to a schedule for consecutively stered doses of an active protein, either alone or as a part of a fusion protein composition, wherein the doses are given in eutically effective amounts to INCORPORATED BY REFERENCE (RULE 20.6) WO 05226 result in sustained beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition.

As used herein the “anti-inflammatory window” is defined as the range of scIL-lO concentrations that produce anti-inflammatory effects on macrophages, while not inducing immunostimulatory effects (on CD8 T cells, NK cells, etc... ). For example, two assays are used in the Examples to define the potencies of those two ivities: l) PBMC cytokine release assay: yields an IC50 value ly in the low picomolar range) for the concentration at which anti-inflammatory effects occur as measured by inhibition of release of pha (TNFq); and 2) MC/9 proliferation assay: yields an ECSO value (usually in the high picomolar to nanomolar range) for the concentration at which immunostimulation s occur.

The ratio in Tables 11 and 12 is the ratio of (MC/9 ECSO) / (PBMC ICSO) values. These two assays represent an approximation of the two types of ties. IL-lO targets cell populations within PBMCs to suppress their release of pro-inflammatory cytokines upon LPS ation, and IL-10 drives the eration of MC/9 cells at concentrations relevant to its immunostimulatory s. There are many other potential assays that may be used to address the anti-inflammatory window size of the molecules of the invention. However, it is understood that both the immunostimulatory and anti-inflammatory s of O occur in a wider number of cell types. scIL- 10.

Human wild-type IL-lO (thL-lO) is a non-covalently linked dimer protein comprising two identical monomer subunits. Each identical monomer subunit of human wild type IL-lO (thL-lO) has the following amino acid sequence (absent the leader sequence): SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFK GYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPC ENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: I) (UniProtKB- P22301[chain 19-178] of IL 10, Interleukin-10, Homosapiens). SEQ ID NO: 1 is also referred to herein as an “unsubstituted IL-lO monomer subunit”. Amino Acid sequences based on SEQ ID NO: 1 that comprise at least one amino acid substitution are referred to herein as “substituted IL-10 monomer subunits”.

The polypeptides of Formula 1 are referred to herein as “scIL-IO” polypeptides and comprise an amino acid sequence arrangement from N—terminus to C-terminus in accordance with Formula 1: INCORPORATED BY REFERENCE (RULE 20.6) (first monomer subuniU-LINKER-(second monomer subunit) Formula 1 n the first monomer subunit, the second monomer subunit or both the first and second monomer subunits may be independently selected from: an unsubstituted IL-10 monomer subunit; or a tuted IL-10 monomer subunit comprising at least one amino acid substitution; and wherein LINKER is any amino acid linker of at least 1-100 amino acids in length. ably, LINKER has a length of between at least 2 amino acid and less than 100 amino acids, such as for example between at least 2 amino acids and less than 75 amino acids, more ably between at least 3 amino acids and less than 50 amino acids, such as for example between at least 4 amino acids and less than 25 amino acids, such as for example between at least 5 amino acids and less than 20 amino acids and even more preferably between at least 6 amino acids and less than 15 amino acids. More preferably, the linker has a length of between at least 3 amino acids and less than 10 amino acids. Most preferably, the linker has a length of 2, 3 7 4, 5, 67 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. Preferably, the linker is a flexible linker. Preferably, the flexible linker ses or ts of the amino acids glycine, asparagine and/or serine. More preferably, the flexible linker comprises or consists of the amino acids glycine and serine. ably the first r subunit and the second monomer subunit of Formula 1 are both unsubstituted IL-IO monomer subunits and each have the amino acid sequence of SEQ ID NO: 1. These peptides are also referred to herein as “unsubstituted scIl-IO”.

Preferably, scIL-IO peptides of Formula 1 comprise at least one amino acid substitution in either the first r subunit of Formula 1, the second monomer subunit of Formula 1, or in both the first and second monomer subunits of Formula 1. These scIl-lO proteins comprising substituted monomer subunits as compared to human wtscIL-IO of SEQ ID NO: 1 are also referred to herein as “scIL-IO variants”.

A preferred scIL-IO e of the invention is referred to herein as “unsubstituted scIL—10 (10aa linker)” and has the following amino acid sequence: QSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFK GYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPC ENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGGGSGGS PGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG INCORPORATED BY REFERENCE (RULE 20.6) WO 05226 YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCE NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 29 3501) or a ce that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to (SEQ ID NO: 2). The ten amino acid linker between the two IL-10 subunits at amino acids 8 is indicated by underlining. It is understood that other ntly linked IL-lO dimer proteins may include any suitable flexible peptide linker and may also be longer or shorter than the underlined sequence of SEQ ID NO: 2. scIL-10 (10aa linker) as represented by SEQ ID NO: 2 comprises two unsubstituted scIL-10 monomer subunits each comprising the amino acid sequence of SEQ ID NO: 1 and as per Formula 1, a LINKER, n LINKER is 10 amino acids in length having the sequence: GGSGGGGSGG (SEQ ID NO: 3). Preferably LINKER of scIL-lO is not SEQ ID NO: 3.

Other preferred unsubstituted scIL-lO peptides of Formula 1 include peptides wherein LINKER is a 5 amino acid linker also referred to herein as “unsubstituted sc-ILlO (Saa linker)”. One preferred five, amino acid linker is the sequence: GGSGG (SEQ ID NO: 4).

Other preferred unsubstituted scIL-lO peptides of Formula 1 include es wherein LINKER is a three amino acid linker also referred to herein as “unsubstituted O (3 aa linker)”. One preferred three, amino acid linker is the sequence is the sequence GGG.

The t invention is based in part on the discovery that fusion proteins comprising unsubstituted scIL-10 as represented by a 1 and scIL-10 comprising at least one amino acid substitution (“scIL-10 variants”) also represented by Formula 1, possess a broad anti- inflammatory window. The present invention is also based in part on the discovery that certain amino acid substitutions of unsubstituted scIL-10 further increase the immunostimulatory ECso. The ability to se the immunostimulatory EC50 while maintaining a low anti-inflammatory ICso provides several orders of ude increase in the anti-inflammatory window size as compared to, for example, wild-type IL—10 or other fusion proteins comprising IL-10 that are not modified in accordance with the invention.

Without being limited to any theory, it is believed that amino acid tutions at the interface of scIL-lO with the IL—lORl and/or IL-lO R2 receptor resulted in modulation of IL- 10’s immunostimulatory properties, anti-inflammatory properties or both.

It was found that an amino acid substitution at aspartic acid at position 41 (based on SEQ ID NO: 1) in the first monomer t or at aspartic acid at position 41 (based on SEQ ID NO: 1) of the second monomer subunit of scIL-10 of Formula 1 disrupts at least one of the scIL-10 interfaces with its IL-10R1 or thereby slightly weakening the anti- INCORPORATED BY REFERENCE (RULE 20.6) inflammatory potency while significantly weakening the immunostimulatory potency of scIL-IO resulting in an increase in the anti-inflammatory . It was also found that mutations that disrupt scIL-lO at one interface with IL-10R1 on one of either the first or second r subunit and also disrupts scIL-lO at one interface with IL-10R2, (for example at the methionine at position 22 of SEQ ID NO: 1) on either the first or second monomer subunit that is not the same as the mutation that disrupts the IL-lO R1 interface provides an extremely large anti-inflammatory window.

It was also discovered that an amino acid substitution of isoleucine at position 87 (based on SEQ ID NO: 1) and which is believed to affect the binding to both IL-IORI and IL- 10R2 appears to have a similar effect as when scIL-IO is designed to disrupt IL-IORI in one t and disrupt IL-10R2 in the other subunit. Without being limited to any theory, it is believed that the isoleucine at position 87 in human thL-lO modulates the interaction with both IL-10 receptors gh it is not clear how such interaction takes place.

Preferably, the invention provides O variants wherein at least one amino acid substitution (as compared to human wild type IL-10 of SEQ ID NO: 1) is introduced in the first and/or second r subunit of Formula 1. Preferably scIL-10 ses at least one amino acid substitution at the interface of the IL-lORl ace on only one of the first or second monomer subunits of Formula 1 but not both of the first or second monomer subunits of Formula 1. Even more preferably scIL-10 comprises at least one amino acid substitution at the interface of the l interface of only one of the first or second monomer subunits of Formula 1 and also comprises at least one amino acid substitution at an IL-10R2 interface on only one of the first or second monomer subunits of Formula that is not the same monomer subunit as the amino acid substitution at the IL-l-Rl interface.

Preferred amino acid substitutions for scIL-10 ts are based on the numbering of amino acids of SEQ ID NO: 1 and include the following mutations: methionine at position 22 and aspartic acid at position 41.

Preferably the invention provides scIL-10 ts wherein at least one amino acid is substituted at position 41 in the first or second monomer subunit of Formula 1 and at least one amino acid is substituted at position 22 in the first or second monomer subunit that is not the same subunit that comprises the amino acid substitution at position 41.

Preferably the ion provides scIL-IO variants wherein at least one amino acid is substituted at the isoleucine at position 87 of only the first monomer subunit or the second monomer t of Formula 1 but not at both monomer subunits.

ORATED BY REFERENCE (RULE 20.6) Amino acid substitutions of methionine at position 22, ic acid at position 41 and isoleucine at position 87 may include substitution With any other amino acid. Either conservative or non-conservative amino acid substitutions can be made at one or more amino acid residues. Both conservative and non-conservative substitutions can be made.

Conservative replacements are those that take place Within a family of amino acids that are related in their side chains. cally encoded amino acids can be divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine, (3) nonpolar (hydrophobic)=cysteine, alanine, valine, e, isoleucine, proline, phenylalanine, methionine, tryptophan, glycine, tyrosine; and (4) uncharged polar=asparagine, glutamine, serine, threonine. Non-polar may be subdivided into: ly hydrophobic=alanine, valine, leucine, isoleucine, methionine, phenylalanine and tely hydrophobic=glycine, e, cysteine, tyrosine, tryptophan. In alternative fashion, the amino acid repertoire can be grouped as (1) acidic=aspartate, glutamate, (2) basic=lysine, arginine, histidine, (3) aliphatic=glycine, alanine, , leucine, isoleucine, , threonine, With serine and threonine ally be grouped separately as aliphatic-hydroxyl, (4) aromatic=phenylalanine, tyrosine, tryptophan, (5) amide=asparagine, glutamine, and (6) sulfur-containing=cysteine and methionine.

Preferred amino acid substitutions for the first monomer subunit and/or the second r subunit in accordance with Formula 1 include the following substitutions: the methionine at position 22 to alanine (M22A); aspartic acid at position 41 to asparagine (D41N); ic acid at position 41 to alanine (D41A); aspartic acid at position 41 to phenylalanine (D41F); isoleucine at position 87 to alanine (I87A).

The invention is also based in part on the discovery that the stimulatory or anti-inflammatory activities of O and scIL-lO variants can be r modulated by fusing scIL-lO or scIL-lO variants to fusion partners including, but not limited to, Fc polypeptides and modified Fc polypeptides such as single chain Fc fusion proteins, mucin linker Fc fusions, Fc polypeptides with truncated hinge regions. Other fusion rs include, but are not limited to: mucin domain polypeptides, albumin fusion proteins, transferrin proteins and other fusion partners not comprising an Fc domain.

INCORPORATED BY REFERENCE (RULE 20.6) 2017/038747 Single Chain Fc Fusion Proteins of sc-ILlO Single chain Fc fusion proteins of the invention have the following arrangement from amino-terminus (N-terminus) to carboxy-terminus (C-terminus) as shown in Formula 2: (scIL-lO)-Ll-HINGE:Fc (Formula 2) wherein, scIL-lO has the amino acid ce of Formula 1, L1 is a linker having the following arrangement from amino-terminus to carboxy-terminus: L2-CL-L3-CH1-L4 (Formula 3) or L2-CH1-L3-CL-L4 (Formula 4) wherein, L2 and L4 are independently polypeptide linkers or are independently absent, L3 is a polypeptide linker; CL is a nt region polypeptide from an immunoglobulin light chain, and CH1 a constant region polypeptide from a CH1 domain of an immunoglobulin heavy chain, HINGE is a hinge sequence of an immunoglobulin or is absent with the proviso that if HINGE is , L4 is present; and Fc is the y-terminus of an immunoglobulin or any active fragment or derivative thereof In accordance with the ion, an scIL-lO of Formula 1 is fused to the N-terminal region of an immunoglobulin Fc region via a novel linker (L1) that is derived from the CL and CH1 domains of an immunoglobulin arranged as a single chain (sc) also ed to herein as “scCLCHl linkers” (Formula 3).

The C-terminus of the CL region may be linked to the N-terminal region of a CH1 region via polypeptide linker L3. The N-terminus of the CL region may be fused to the C- terminus of scIL-lO of Formula 1 via an optional polypeptide linker L2. The C-terminus of the CH1 domain is linked to the Fc domain via an immunoglobulin hinge region (HINGE) or a ptide linker (L4) or both a hinge (HINGE) and a polypeptide linker (L4).

The C-terminus of the CH1 domain may also be linked to the N-terminus of a CL region via polypeptide linker L3. The N-terminus of the CH1 region may be fused to the C- terminus of scIL-lO of Formula 1 via an optional polypeptide linker L2. The C-terminus of the CL region is linked to the Fc region via an immunoglobulin hinge region (HINGE) or a ptide linker (L4) or both a hinge (HINGE) and a polypeptide linker (L4).

INCORPORATED BY REFERENCE (RULE 20.6) Preferably, L3 is selected from ial flexible domains sing amino acids selected from Gly (G), and/or Ser (S). Preferably, the linker is comprised of polypeptide of the general formula ly-Gly-Ser (SEQ ID NO: 5))n or (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 6))n wherein n is an integer from I to 10. Preferably, each linker is a polypeptide comprising from about 1 to about 100 amino acids, preferably about 1-50 amino acids, preferably about 1-25 amino acids, ably about 1-15 amino acids preferably about 1-10 amino acids, preferably about 4-24 amino acids, preferably about 5-20 amino acids preferably about 5-15 amino acids and preferably about 5-10 amino acids. Preferably, the linker is (Gly- Gly-Gly-Gly-Ser (SEQ ID NO: 6)) n wherein n is 2 or 4.

L2 and L4 are independently selected from artificial flexible domains comprising amino acids selected from, for example, Gly (G), and Ser (S). Preferably, the linker is comprised of polypeptide of the general formula (Gly-Gly-Gly-Ser (SEQ ID NO: 5))n or (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 6))n wherein n is an integer from I to 10. Preferably, each linker is a polypeptide comprising from about 1 to about 100 amino acids, preferably about 1-50 amino acids, preferably about 1-25 amino acids, preferably about 1-15 amino acids ably about 1-10 amino acids, preferably about 4-24 amino acids, preferably about -20 amino acids preferably about 5-15 amino acids and preferably about 5-10 amino acids.

Preferably, the linker is (Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 6))n wherein n is 2 or 4.

L2, L3 and L4, may further comprise amino acids such as, for example, Lys (K), Thr (T), Glu (E), and Asp (D).

The CL region of the novel scCLCHl linker (L1) may be substantially identical to the ponding CL region of native immunoglobulins belonging to any of the immunoglobulin s, i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG dy subclasses, i.e., IgGl, IgG2, IgG3, and IgG4. The CL region (Ll) may have amino acid sequence that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding CL region of native immunoglobulins belonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e., IgGl, IgG2, IgG3, and IgG4. If the CL region of L1 is a modified derivative or variant of a native CL region such modifications include, but are not limited to, amino acid insertions, deletions, substitutions and ngements. Preferably, the amino acid sequence of the CL region in accordance with the invention, is at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% identical to the ponding CL region of native immunoglobulins belonging to any of the globulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e., IgGl, IgG2, IgG3, and IgG4.

INCORPORATED BY REFERENCE (RULE 20.6) The CHl region of the novel scCLCHl linker (Ll) may be substantially identical to the corresponding CHl region of native immunoglobulins belonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e., IgGl, IgG2, IgG3, and IgG4. The CH1 region of L1 may have amino acid sequence that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding CH1 region of native immunoglobulins belonging to any of the globulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e., IgGl, IgG2, IgG3, and IgG4. If the CH1 region of the L1 linker is a modified derivative or variant of a native CH1 immunoglobulin region such modifications include, but are not limited to, amino acid insertions, deletions, substitutions and rearrangements.

Preferably, the amino acid sequence of the CH1 region is at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% identical to the corresponding CH1 region of native immunoglobulins belonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e., IgGl, IgG2, IgG3, and IgG4.

The CH1 region and CL regions of L1 do not need to be identical to or a variant of, the corresponding regions of the same immunoglobulin class. For example, the CL region may be derived from the corresponding region of IgE and the CH1 region may be derived from the corresponding region of IgG.

Preferably, CL and CH1 0f the scCLCHl linker are derived from the ponding CL and CH1 s of IgG1, preferably human IgG1.

An exemplary CL region corresponding to the CL region of a human IgG] (hIgGl) includes: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES (SEQ ID NO: 7).

An exemplary CH1 region corresponding to the CH1 region of hIgGl includes: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 8).

The single chain Fc fusion proteins disclosed herein comprise an Fc region that es at least a portion of the carboxy-terminus of an immunoglobulin heavy chain. For example, the Fc portion may comprise: a CH2 domain, a CH3 domain, a CH4 domain, a CH2-CH3 domain, a CH2-CH4 domain, a 3-CH4 domain, a CH2 , a hinge-CH2-CH3 , a hinge-CH2-CH4 , or a hinge-CH2-CH3-CH4 domain. The INCORPORATED BY REFERENCE (RULE 20.6) Fc domain may be derived from antibodies ing any of the immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e., IgGl, IgG2, IgG3, and IgG4. Preferably, the Fc region is derived from IgGl preferably human IgG1.

The Fc domain may be a naturally occurring Fc sequence belonging any of the immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e., IgGl, IgG2, IgG3, and IgG4, including l allelic or splice variants.

Alternatively, the Fc domain may be a hybrid domain comprising a portion of an Fc domain from two or more different Ig es, for example, an IgG2/IgG4 hybrid Fc domain.

Preferably, the Fc domain is derived from a human immunoglobulin molecule. Alternatively, the Fc domain may be a humanized or deimmunized (removal of T cell epitopes which can activate helper T cells) version of an Fc domain from a non-human animal, including but not limited to mouse, rat, rabbit, and monkey.

The Fc domain may be a variant Fc sequence, e. g., an Fc sequence that has been modified (e.g., by amino acid tution, on and/or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural features and/or biological activity. For example, one may make cations in the Fc region in order to generate an Fc variant that (a) has increased or decreased antibody-dependent ediated cytotoxicity (ADCC), (b) increased or decreased complement mediated cytotoxicity (CDC), (0) has increased or decreased affinity for Clq and/or (d) has increased or decreased affinity for a Fc receptor relative to the parent Fc. Such Fc region ts will generally se at least one amino acid modification in the Fc . Combining amino acid modifications is thought to be particularly desirable. For example, the variant Fc region may include two, three, four, five, etc. substitutions therein, e. g. of the specific Fc region ons identified .

The hinge region of the Fc fusion proteins of the invention may be derived from antibodies belonging to any of the immunoglobulin s, i.e., IgA, IgD, IgE, IgG, or IgM.

The hinge region may be derived from any of the IgG antibody sses, i.e., IgGl, IgG2, IgG3, and IgG4. The hinge region may naturally contain a cysteine residue or may be engineered to contain one or more cysteine residues.

Preferably, the hinge region may have an amino acid sequence that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding hinge region of native immunoglobulins belonging to any of the immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e., IgGl, IgG2, IgG3, and IgG4. Preferably, the amino acid sequence of the hinge region is at least 80%, more INCORPORATED BY REFERENCE (RULE 20.6) preferably at least 85%, more preferably at least 90%, and more preferably at least 95% identical to the corresponding hinge region of human IgG1.

Shown below is the sequence of a human IgG1 globulin constant region, and the relative position of the hinge region is indicated by solid underlining: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9). The CHI region is ted by ining With a dotted line, and the CH2 and CH3 regions are indicated by bold lettering. The C-terminal lysine of an IgG sequence may be removed or replaced With a non-lysine amino acid, such as e, to further increase the serum half-life of the Fc fusion protein.

The hinge sequence may include substitutions that confer desirable pharmacokinetic, biophysical, and/or biological properties. An exemplary hinge region of the ion comprises an amino acid sequence that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the following: EPKSSDKTHTCPPCP (SEQ ID NO: 51).

The Fc domain and the hinge region may be derived from one antibody class or subclass. For example, the hinge region and the Fc domain may be derived from IgG]. The Fc domain and hinge region may correspond to different antibody classes or subclasses. For example, the Fc domain may correspond to the Fc region of IgG2 or IgG4 and the hinge region may correspond to IgGl.

Preferably, all immunoglobulin domains of the Fc fusion proteins of the invention are derived from IgGl, preferably human IgG1. Preferably, the combined hinge region and Fc region of the fusion proteins of the invention comprise an amino acid sequence that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 10). Preferably, the combined hinge region and Fc region of the fusion ns of the ion se an amino acid sequence that is at least 50%, 60%, INCORPORATED BY REFERENCE (RULE 20.6) 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to: EPKSQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQV KFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQleLQGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 11).

It may be desirable to have a hinge sequence and/or Fc region of the single chain fusion proteins of the invention comprising a free cysteine residue in order to permit the formation of a disulfide bond n the hinge and or PC regions thereby g dimers of the Fc fusion proteins of the invention. It may be desirable to alter the hinge and/or Fc region ces to remove free cysteine es, e. g., by ng one or more cysteine residues in a linker to another residue, such as a serine, alanine or glycine. The hinge region of the single chain fusion ns of the invention may comprise one or more free cysteine es capable of forming one or more disulfide bonds with a second single chain fusion protein of the invention thereby forming a dimer complex. ably, the (scIL-lO)—Ll-HINGE-Fc fusion proteins of the invention are dimer complexes comprising two monomeric single chain (scIL-10)-L1-HINGE-Fc fusion ns of the invention linked via a disulfide bond to the hinge region or in the Fc region of the other r. The dimer complexes may be homodimeric (e.g. both monomeric fusion proteins are identical) or heterodimeric (e. g. O may be different for each monomeric fusion protein). Preferably, the dimer complexes are homodimers thereby forming a homodimeric complex that provides an antibody configuration that resembles that of a native antibody.

Without being limited to any one theory, it is believed that the homodimeric fusion proteins of the invention se half-life due to the presence of a dimerized Fc region which more closely resembles the native antibody structure as compared to traditional Fc fusion proteins. This is particularly true when the fusion protein has the configuration of Formula 3.

A more native Fc domain antibody configuration is believed to enable better binding to the FcRn receptor and therefore increase the circulating half-life of the of the scIL-lO-Ll- HINGE-Fc dimer complex.

Another improved property associated with scIL-lO-Ll-HINGE-Fc dimer complexes is that bioactivity is increased versus a traditional Fc fusion proteins based on the use of the scCLCHl linker which imparts flexibility to relieve steric hindrance caused by the dimerization through the Fc in the hinge region.

INCORPORATED BY REFERENCE (RULE 20.6) WO 05226 Preferably the invention provides (scIL-10)-L1-HINGE-Fc fusion wherein scIL-10 of Formula 1 is tituted scIL-10 (10aa linker). Preferably the invention provides (scIL- -HINGE-Fc fusion wherein the scIL-10 of Formula 1 is an 10 variant comprising at least one amino acid substitution in the first monomer subunit or the second monomer subunit as per a 1 selected from the methionine at position 22, the ic acid at position 41, and the isoleucine at position 87 or any combination thereof. Preferably there is at least one amino acid substitution at position 41 in the first or second r subunit of Formula 1 and at least one amino acid is substituted at position 22 in the first or second monomer subunit that is not the same t that comprises the amino acid substitution at on 41.

A preferred scILlO-Ll-HINGE-Fc fusion protein of the invention comprises an amino acid sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12 wherein scIL-10 is unsubstituted scIL-10 (10aa ).

Preferred O-Ll-HINGE-Fc fusion proteins of the invention comprise an amino acid sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 20-21 and 37-44 all as shown in Table 4.

Preferred O-Ll-HINGE-Fc fusion ns of the invention comprise an amino acid sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 17-19 as shown in Table 4 wherein scIL-lO is an scIL-10 variant.

The invention also provides nucleic acids encoding any of the various fusion proteins disclosed herein. Codon usage may be selected so as to improve expression in a cell. Such codon usage will depend on the cell type selected. Specialized codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See for example: Mayfield et al., Proc. Natl. Acaa’. Sci. USA, 100(2):438-442 (Jan. 21, 2003); Sinclair et al., Protein Expr. Purif, 26(I):96-105 (October 2002), Connell, N.D., Curr. Opin. Biotechnol, 12(5):446-449 (October 2001), Makrides et al.,Microbiol Rev, 60(3):512-538 (September 1996), and Sharp et al., Yeast, 57-678 (October 1991).

General techniques for nucleic acid manipulation are described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), or Ausubel, F. et al., Current Protocols in Molecular Biology, Green Publishing and Wiley-Interscience, New York (1987) and periodic updates, herein incorporated by reference. Generally, the DNA encoding the polypeptide is operably linked to suitable transcriptional or translational regulatory elements derived from INCORPORATED BY REFERENCE (RULE 20.6) WO 05226 mammalian, viral, or insect genes. Such regulatory elements include a riptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants is additionally incorporated.

The fusion proteins described herein may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is ably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal ase) by the host cell. An exemplary N-terminal leader sequence for production of polypeptides in a mammalian system is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 48), which is removed by the host cell following expression.

For prokaryotic host cells that do not ize and process a native signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, llinase, or heat-stable enterotoxin II leaders.

For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, a factor leader (including Saccharomyces and Kluyveromyces alpha-factor leaders), or acid phosphatase , the C. albicans glucoamylase , or the signal described in US. Pat. No. 5,631,144. In ian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA for such precursor regions may be ligated in reading frame to DNA encoding the protein.

Both expression and cloning vectors contain a nucleic acid ce that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that s the vector to replicate independently of the host chromosomal DNA, and es origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and s. The origin of replication from the plasmid pBR322 is suitable for most egative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (SV40, a, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the INCORPORATED BY REFERENCE (RULE 20.6) origin of replication component is not needed for ian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e. g., ampicillin, neomycin, methotrexate, or tracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not ble from complex media, e. g., the gene encoding D-alanine racemase for i. sion and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the n disclosed herein, e.g., a fibronectin-based scaffold protein. Promoters suitable for use with prokaryotic hosts include the phoA promoter, beta-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tan promoter. r, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgamo (S.D.) sequence operably linked to the DNA encoding the protein disclosed . Promoter sequences are known for eukaryotes.

Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where ription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT (SEQ ID NO: 49) region where N may be any nucleotide. At the 3’ end of most eukaryotic genes is an AATAAA (SEQ ID NO: 50) sequence that may be the signal for addition of the poly A tail to the 3’ end of the coding sequence. All of these sequences are ly inserted into eukaryotic expression vectors. es of suitable promoting sequences for use with yeast hosts e the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehydephosphate dehydrogenase, nase, pyruvate decarboxylase, phosphofructokinase, glucosephosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Transcription from vectors in mammalian host cells can be controlled, for e, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian a virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g, the actin er or an immunoglobulin promoter, from hock promoters, provided such promoters are compatible with the host cell systems.

INCORPORATED BY REFERENCE (RULE 20.6) Transcription of a DNA encoding proteins disclosed herein by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, n, protein, and n). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication , and adenovirus enhancers. See also Yaniv, Nature, 297: 17-18 (1982) on ing ts for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5’ or 3’ to the peptide-encoding sequence, but is preferably located at a site 5’ from the promoter.

Expression vectors used in eukaryotic host cells (e. g., yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5’ and, occasionally 3’, slated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of mRNA encoding the protein disclosed herein. One useful ription termination component is the bovine growth e polyadenylation region. See W0 94/ 1 1026 and the expression vector disclosed therein.

The recombinant DNA can also include any type of protein tag ce that may be useful for purifying the protein. Examples of protein tags include but are not limited to a histidine tag, a FLAG tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning and sion vectors for use with bacterial, fungal, yeast, and ian cellular hosts can be found in Cloning Vectors." A Laboratory Manual, (Elsevier, New York (1985)), the relevant disclosure of which is hereby incorporated by reference.

The expression construct is introduced into the host cell using a method appropriate to the host cell, as will be apparent to one of skill in the art. A variety of methods for ucing c acids into host cells are known in the art, including, but not limited to, electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; rojectile bombardment, lipofection, and infection (where the vector is an infectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, or ial cells.

Suitable bacteria include gram negative or gram ve organisms, for example, E. coli or Bacillus spp. Yeast, preferably from the Saccharomyces species, such as S. cerevisiae, may INCORPORATED BY REFERENCE (RULE 20.6) also be used for production of polypeptides. Various mammalian or insect cell culture systems can also be employed to express recombinant ns. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow et al.

(Bio/Technology, 6:47 (1988)). Examples of suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines. ed polypeptides are ed by culturing suitable host/vector systems to express the recombinant ns. For many ations, the small size of many of the polypeptides disclosed herein would make expression in E. coli as the preferred method for expression.

The protein is then purified from culture media or cell extracts.

In other aspects, the invention provides host cells containing vectors encoding the fusion proteins described herein, as well as methods for ing the fusion proteins described herein. Host cells may be transformed with the herein-described expression or cloning vectors for protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Host cells useful for high-throughput protein production (HTPP) and ale production include the HMS l74-bacterial strain. The host cells used to e the proteins disclosed herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640 ), and Dulbecco's ed Eagle's Medium ((DMEM), Sigma)) are le for culturing the host cells. In addition, many of the media described in various scientific literature may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or mal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), tides (such as adenosine and thymidine), antibiotics (such as Gentamycin drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy . Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those usly used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The fusion proteins provided herein can also be produced using cell-translation systems. For such purposes the nucleic acids encoding the fusion protein must be modified to allow in vitro ription to produce mRNA and to allow cell-free translation of the mRNA INCORPORATED BY REFERENCE (RULE 20.6) in the ular cell-free system being utilized (eukaryotic such as a mammalian or yeast cell-free translation system or prokaryotic such as a bacterial ree translation system).

The fusion proteins disclosed herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd Edition, The Pierce Chemical Co, rd, Ill. (1984)). Modifications to the fusion proteins can also be produced by chemical synthesis.

The fusion proteins disclosed herein can be purified by isolation/purification methods for proteins generally known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, g out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reversed- phase chromatography, get filtration, gel permeation chromatography, affinity chromatography, electrophoresis, rcurrent distribution or any combinations of these.

After purification, polypeptides may be exchanged into different buffers and/or concentrated by any of a variety of methods known to the art, including, but not limited to, filtration and dialysis.

The d fusion protein is preferably at least 85% pure, or ably at least 95% pure, and most preferably at least 98% pure. less of the exact numerical value of the purity, the fusion protein is sufficiently pure for use as a pharmaceutical product.

Other Fusion Partners.

Other appropriate fusion partners for scIL-lO proteins of the invention include but are not limited to proteins comprising an Fc region of all other types.

For example, scIL-10 proteins may be fused ly to the hinge region of a native immunoglobulin containing an Fc , for example IgGl. SEQ ID NO: 13 is an example of unsubstituted scIL-lO (5aa linker) fused to the hinge region of an IgGl molecule. The IgGl molecule may be modified, by, for e, by shortening the hinge region of IgG1.

SEQ ID NO: 14 is an example of scIL-lO (5aa linker) fused to the hinge region of IgG1 wherein in the hinge region of the native IgGl has been shortened by 4 amino acids. SEQ ID NO: 15 is an e of scIL-lO fused to the hinge region of IgG1 wherein in the hinge region of the native IgGl has been shortened by 7 amino acids. SEQ ID NO: 16 is an example of scIL-lO fused to the hinge region of IgG1 n in the hinge region of the native IgGl has been shortened by 10 amino acids.

INCORPORATED BY REFERENCE (RULE 20.6) A preferred fusion partner comprises an Fc region further comprising a mucin-domain polypeptide linker as is described in WO2013/184938 incorporated herein by nce. A “mucin-domain polypeptide linker” is defined herein as any protein comprising a “mucin domain” capable of being linked to one or more fusion polypeptide rs. A mucin domain is rich in ial ylation sites, and has a high t of serine and/or threonine and proline, which can ent greater than 40% of the amino acids within the mucin domain. A mucin domain is heavily glycosylated with predominantly O-linked glycans. A mucin-domain polypeptide has at least about 60%, at least 70%. at least 80%, or at least 90% of its mass due to the glycans. Mucin domains may comprise tandem amino acid repeat units (also referred to herein as TR) that may vary in length from about 8 amino acids to 150 amino acids per each tandem repeat unit. The number of tandem repeat units may vary between 1 and 25 in a mucin-domain polypeptide of the invention.

Mucin-domain polypeptide linkers of the invention include, but are not limited to, all ’ is meant that the mucin polypeptide or a portion of a mucin protein. A “portion thereof linker comprises at least one mucin domain of a mucin protein. Mucin proteins e any protein encoded for by a MUC gene (e.g., MUC], MUC2, MUC3A, MUC3B, MUC4, MUCSAC, MUCSB, MUC6, MUC7, MUC8, MUC9, MUCll, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC20, MUC21). The mucin domain of a mucin protein is typically flanked on either side by non-repeating amino acid regions. A mucin-domain polypeptide may comprise all or a portion of a mucin protein (e. g. MUC20). A mucin- domain polypeptide may comprise all or a portion of a mucin n of a soluble mucin protein. Preferably the domain polypeptide ses the extracellular portion of a mucin protein.

Preferably, an scIL-lO protein of Formula 1 is covalently linked to a molecule comprising an Fc region via a mucin-domain polypeptide linker. SEQ ID NO: 52 is an example of unsubstituted scIL-lO fused to mucin linker which is in turn fused to the hinge of a native IgGl Fc region.

A preferred fusion partner is a mucin domain ptide (not including an Fc region) as is described in W0 2013/184939.

A preferred fusion partner comprises serum albumin or a domain of serum albumin.

Human serum albumin is preferred when the fusion proteins of the invention are used for ng humans. In another embodiment, fusion partners comprise human transferrin.

INCORPORATED BY REFERENCE (RULE 20.6) Uses of scIL-lO proteins In one aspect, the invention provides scIL-lO (including fusions of scIL-IO to an appropriate fusion partner and dimerized complexes thereof) that are useful as diagnostic or therapeutic agents. In one aspect, the invention provides proteins useful in the treatment of ers.

The invention also es a method for achieving a beneficial effect in a subject comprising the step of administering to the t a therapeutically or lactically- effective amount of scIL-IO (including fusions of scIL-IO to an appropriate fusion partner and dimerized complexes thereof) of the invention. The effective amount can e a beneficial effect in helping to treat a disease or disorder. In some cases, the method for achieving a beneficial effect can include administering a therapeutically effective amount of a fusion protein composition to treat a subject for diseases and disease categories wherein a therapeutic protein or peptide does not exist.

Preferably scIL-IO is not linked to any fusion partner.

Preferably, scIL-lO is covalently linked to an appropriate fusion partner such as scIL- lO-LI-HINGE-Fc. Preferably, the invention provides dimer complexes of scIL-IO fused to an appropriate fusion partner. ably scIL-IO (including fusions of scIL-IO to an riate fusion r and dimerized complexes thereof) are used to treat patients who suffer from, for example, autoimmune disorders, fibrotic es, inflammatory diseases, ischemic diseases, neurodegenerative diseases, neuropathic diseases, pain disorders, ry disorders, atric disorders, cancer and trauma and injury.

Examples of autoimmune disorders include, but are not d to: acute disseminated encephalomyelitis , acute necrotizing hemorrhagic leukoencephalitis, n’s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid me (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative me (ALPS), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroiditis, autoimmune urticaria, axonal & neuronal neuropathies, Balo disease, Behcet’s e, myopathy, Castleman disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), INCORPORATED BY REFERENCE (RULE 20.6) cicatricial goid/benign mucosal pemphigoid, Cogans syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, Crohn's disease, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), discoid lupus, Dressler’s syndrome, endometriosis, eosinophilic esophagitis, philic fasciitis, ma nodosum, essential mixed cryoglobulinemia, Evans syndrome, experimental allergic alomyelitis, yalgia, fibrosing alveolitis, giant cell arteritis (temporal tis), giant cell myocarditis, glomerulonephritis, sture’s syndrome, granulomatosis with Polyangiitis (GPA) (formerly called Wegener’s omatosis), Grave's disease, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic ary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, le es (Type 1 diabetes), juvenile myositis, Kawasaki e, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen , lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), Lupus (systemic lupus matosus), Lyme disease, chronic, Meniere’s disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha—Habermann disease, multiple sclerosis (MS), myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic’s), neutropenia, ocular cicatricial goid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis), Parsonnage-Tumer syndrome, pemphigus, peripheral neuropathy, perivenous alomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary y cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia, ma gangrenosum, Raynauds phenomenon, reactive Arthritis, reflex hetic dystrophy, Reiter’s syndrome, relapsing ondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis (RA), toid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren’s me, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis, Susac’s syndrome, sympathetic ophthalmia, Takayasu’s arteritis, Temporal arteritis/Giant cell arteritis, thrombocytopenic purpura, Tolosa—Hunt syndrome, transverse myelitis, type 1 diabetes, type I, II, & III autoimmune polyglandular syndromes, INCORPORATED BY REFERENCE (RULE 20.6) WO 05226 2017/038747 ulcerative colitis, undifferentiated tive tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener’s omatosis. es of fibrotic diseases Which may be treated by the scIL-lO and scIL-lO variant peptides (including fusions of each to an appropriate fusion partner) of the invention include, but are not limited to: ve capsulitis, arthroflbrosis, atrial fibrosis, c kidney disease, cirrhosis of the liver, cystic fibrosis (CF), ren's contracture, endomyocardial fibrosis, glial scar, idiopathic pulmonary fibrosis, , macular degeneration, mediastinal fibrosis, myelofibrosis, NAFLDWASH, nephrogenic systemic fibrosis, Peyronie's disease, progressive massive fibrosis (lungs), proliferative vitreoretinopathy, pulmonary fibrosis, retroperitoneal is, scar tissue formation resulting from strokes, scleroderma, systemic sclerosis, tissue adhesion.

Examples of inflammatory diseases include, but are not limited to: allergic enteritis, alpha-l-antitrypsin deficiency, ankylosing spondylitis, asthma, Barrett's esophagus, Behcet's disease, chronic fatigue me (CFS / CFIDS / ME), chronic Lyme disease (borreliosis), cocaine-associated vasculitis, Crohn's disease, deficiency of the Interleukin-l Receptor Antagonist (DIRA), sion, diabetes al Mediterranean Fever (FMF), fibromyalgia (FM), gastroesophageal reflux disease (GERD), glomerulonephritis, graft versus host disease, granulomatous angiitis, Hashimoto's thyroiditis, hypertension, hyperthyroidism, hypothyroidism, inflammatory bowel disease (IBD), inflammatory myopathies (polymyositis, inclusion body myositis, dermatomyositis), interstitial cystitis (IC), irritable bowel syndrome (IBS), ischemic colitis, kidney stones, Lofgren's syndrome, Lupus erythematosis, phetamine-associated vasculitis, ne headache, Morgellon's, multiple chemical ivity (MCS), multiple sis (MS), neonatal onset multisystem inflammatory disease (NOMID), optic neuritis, osteoarthritis, pemphigus vulgaris, polymyalgia rheumatica, prostatitis, psoriasis, psoriatic arthritis, radiation colitis, Raynaud's syndrome/phenomenon, reactive arthritis (Reiter me), reflex sympathetic dystrophy (RSD), restless leg syndrome, rheumatoid arthritis (RA), sarcoidosis, scleroderma, seasonal affective disorder (SAD), septic shock, sinusitis, SjOgren's syndrome, temporal tis, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), tive colitis, uveitis, vasculitis, and vertigo.

Examples of ischemic diseases include, but are not limited to: acute coronary syndrome, angina pectoris, angor animi, copeptin, coronary artery disease, coronary ischemia, hibernating myocardium, ischemic , management of acute coronary syndrome, meldonium, myocardial infarction, dial infarction complications, INCORPORATED BY REFERENCE (RULE 20.6) dial infarction diagnosis, myocytolysis, post-anoxic encephalopathy, Prinzmetal's angina, Sgarbossa's criteria, stroke, TIMI, ent ischemic attack (TIA) and unstable angina.

Examples of neurodegenerative diseases include, but are not limited to: ataxia telangiectasia, autosomal dominant cerebellar ataxia, —Yoshinari syndrome, Batten disease, estrogen and neurodegenerative es, hereditary motor and sensory neuropathy with proximal dominance, Infantile Refsum disease, JUNQ and IPOD, locomotor ataxia, Lyme disease, Machado—Joseph disease, mental retardation and microcephaly with pontine and cerebellar hypoplasia, multiple system atrophy, neuroacanthocytosis, neuronal ceroid lipofuscinosis, Niemann—Pick disease, pontocerebellar hypoplasia, protein aggregation, pyruvate dehydrogenase deficiency, radiation myelopathy, Refsum e, retinitis pigmentosa, Sandhoff disease, Shy-Drager syndrome, spinal muscular atrophy, erebellar ataxia, subacute combined degeneration of spinal cord, subacute sclerosing panencephalitis, Tabes dorsalis, Tay—Sachs disease, toxic encephalopathy, toxic leukoencephalopathy and Wobbly og Syndrome.

Examples of neuropathic diseases include, but are not limited to: Bell's Palsy, campylobacter-associated motor axonopathies, t-Marie-Tooth, c inflammatory demyelinating uropathy, diabetic amyotrophy avulsion, diabetic neuropathies, Guillain Barre Syndrome and vasculitis.

Examples of pain disorders include, but are not limited to: Amplified musculoskeletal pain syndromes, Anterior cutaneous nerve entrapment syndrome, central pain syndrome, chronic functional abdominal pain, chronic pain, chronic prostatitis/chronic pelvic pain me, c wound pain, degenerative disc e, dentomandibular sensorimotor dysfunction, failed back syndrome, fibromyalgia, interstitial cystitis, irritable bowel syndrome (IBS), cial pain syndrome, pelvic pain, post-vasectomy pain syndrome, reflex neurovascular dystrophy, sickle-cell disease, theramine, and ynia.

Examples of auditory disorders include, but are not limited to: tive hearing loss, sensorineural hearing loss (SNHL), mixed hearing loss.

Examples of psychiatric disorders include, but are not limited to: major depressive disorder, treatment-refractory depression, treatment-resistant depression.

Examples of trauma and injury include, but are not d to: ing central nervous system (CNS) injuries, traumatic brain injury, spinal cord injury, crush injuries, shock, tendon damage, wounds to the cornea, wounds to the eye, skin wounds.

INCORPORATED BY REFERENCE (RULE 20.6) Preferably, an scIL-lO proteins (including fusions of scIL-lO to an appropriate fusion partner and dimerized complexes thereof) of the invention may be used to treat ts who suffer from, for e, autoimmune disorders including autoimmune lymphoproliferative syndrome (ALPS), autoimmune thyroiditis, Crohn's e, Grave's disease, Hashimoto's thyroiditis Kawasaki disease, Lupus (systemic lupus erythematosus), multiple sis (MS), myasthenia gravis, psoriasis, rheumatoid arthritis, Sjogren's syndrome, type 1 diabetes, ulcerative colitis, fibrotic es including c Kidney Disease, cirrhosis of the liver, macular degeneration, NASH, proliferative vitreoretinopathy, ary fibrosis, scar tissue formation resulting from strokes, tissue adhesion; including inflammatory diseases including allergic enteritis, alpha-l-antitrypsin deficiency, asthma, Behcet's disease, cocaine- associated vasculitis, glomerulonephritis, Graft Versus Host Disease, granulomatous angiitis, inflammatory bowel disease, inflammatory myopathies (polymyositis, inclusion body myositis, dermatomyositis), ischemic colitis, methamphetamine-associated vasculitis, optic neuritis, gus vulgaris, radiation colitis, sarcoidosis, Septic Shock, temporal arteritis, vasculitis, ischemic diseases including myocardial infarction, post-anoxic encephalopathy, stroke; neurodegenerative diseases including al ceroid lipofuscinosis, radiation myelopathy, retinitis tosa, spinal muscular atrophy; neuropathic diseases including campylobacter-associated motor axonopathies, Charcot-Marie-Tooth, chronic inflammatory demyelinating polyneuropathy, diabetic amyotrophy avulsion, diabetic neuropathies, Guillain Barre Syndrome; auditory disorders including Conductive hearing loss, Sensorineural hearing loss (SNHL), Mixed hearing loss; psychiatric disorders including major depressive disorder, treatment-refractory depression, ent-resistant depression; trauma and injury including central nervous system (CNS) injuries, traumatic brain , spinal cord injury, crush injuries, shock, tendon damage, wounds to the cornea, wounds to the eye, skin wounds.

Most preferably, scIL-lO proteins (including fusions of scIL-lO to an appropriate fusion partner and zed complexes thereof) in accordance with the invention may be used to treat patients who suffer from, for e, autoimmune disorders including mune lymphoproliferative syndrome (ALPS), autoimmune thyroiditis, Crohn's e, Grave's disease, Hashimoto's thyroiditis ki disease, Lupus (systemic lupus erythematosus), multiple sclerosis (MS), enia gravis, psoriasis, rheumatoid arthritis, Sjogren's syndrome, type 1 diabetes, ulcerative colitis; flbrotic diseases including Chronic Kidney Disease, cirrhosis of the liver, macular degeneration, NAFLD/NASH, proliferative vitreoretinopathy, pulmonary fibrosis, scar tissue formation resulting from strokes, tissue adhesion, inflammatory diseases including allergic enteritis, l-antitrypsin deficiency, INCORPORATED BY REFERENCE (RULE 20.6) asthma, Behcet's disease, cocaine-associated vasculitis, glomerulonephritis, Graft Versus Host Disease, granulomatous angiitis, atory bowel disease, inflammatory myopathies (polymyositis, inclusion body myositis, dermatomyositis), ischemic colitis, methamphetamine-associated itis, optic neuritis, pemphigus vulgaris, radiation colitis, sarcoidosis, Septic Shock, temporal arteritis, vasculitis, ischemic diseases including myocardial infarction, post-anoxic encephalopathy, stroke; neurodegenerative diseases ing neuronal ceroid lipofuscinosis, radiation myelopathy, retinitis pigmentosa, spinal ar atrophy, neuropathic diseases including campylobacter-associated motor axonopathies, Charcot-Marie-Tooth, chronic atory demyelinating polyneuropathy, diabetic amyotrophy avulsion, diabetic neuropathies, Guillain Barre Syndrome; auditory disorders including Conductive g loss, Sensorineural g loss (SNHL), Mixed hearing loss, psychiatric disorders including major depressive disorder, treatment-refractory sion, treatment-resistant depression, trauma and injury including central s system (CNS) injuries, traumatic brain injury, spinal cord injury, crush injuries, shock, tendon damage, wounds to the cornea, wounds to the eye, skin wounds.

Preferably scIl-lO proteins (including fusions of scIL-IO to an appropriate fusion partner and dimerized complexes thereof) of the invention may be used to treat patients who suffer from, for example cancer of the uterus, cervix, breast, ovaries, prostate, testes, penis, gastrointestinal tract, esophagus, rynx, stomach, small or large intestines, colon, or rectum, kidney, renal cell, bladder, bone, bone marrow, skin, head or neck, skin, liver, gall bladder, heart, lung, pancreas, salivary gland, adrenal gland, thyroid, brain, s, ganglia, central nervous system (CNS) and peripheral nervous system (PNS), and immune system, spleen or thymus, papilloma virus-induced s, epithelial cell cancers, elial cell cancers, squamous cell carcinomas, adenocarcinomas, carcinomas, melanomas, as, teratocarcinomas, immunogenic tumors, munogenic tumors, dormant , mas, leukemias, as, chemically-induced cancers, metastasis, and angiogenesis, and Tuberous sclerosis.

Preferably, sclL-lO fusion proteins (including fusions of scIL-lO to an appropriate fusion partner and dimerized complexes thereof) in accordance with the invention may be used to treat patients who suffer from auditory disorders, renal cell carcinoma, ma, psoriasis, fibrosis, depression, and inflammatory bowel disease (IBD).

Preferably, sclL-lO fusion ns (including fusions of scIL-lO to an riate fusion partner and dimerized complexes thereof) in accordance with the invention may also be used in the manufacture of a medicament to treat patients to diseases as set forth above, INCORPORATED BY REFERENCE (RULE 20.6) auditory disorders, auditory disorders, renal cell oma, melanoma, psoriasis, fibrosis, depression, and inflammatory bowel disease (IBD).

The application further provides pharmaceutically acceptable itions comprising scIL-lO proteins (including fusions of scIL-10 to an appropriate fusion partner and zed complexes thereof) bed herein. Therapeutic formulations comprising sclL—lO proteins are prepared for storage by mixing the described proteins having the desired degree of purity with al physiologically acceptable carriers, excipients or izers (Remington's ceutical Sciences 16th n, Osol, A. Ed. (1980)), in the form of aqueous solutions, lyophilized or other dried formulations. Acceptable carriers, excipients, or stabilizers are nontoxic to ents at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl um chloride, thonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol; cyclohexanol, 3-pentanol, and m-cresol), low lar weight (less than about 10 residues) polypeptides, ns, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or ol; salt-forming counter-ions such as sodium; metal complexes (e. g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).

The formulations herein may also n more than one active compounds as necessary for the particular indication being treated, ably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The Preferably, scIL-lO proteins (including fusions of scIL-10 to an appropriate fusion partner and dimerized complexes thereof) in accordance With the invention may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, mes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical es 16th edition, Osol, A. Ed. (1980).

INCORPORATED BY REFERENCE (RULE 20.6) The ations to be used for in viva administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable es of solid hydrophobic polymers containing the fibronectin based scaffold proteins described herein, which matrices are in the form of shaped articles, e.g., films, or microcapsules. es of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides, copolymers of lactide and glycolide, copolymers of L-glutamic acid and y ethyl-L-glutamate, gradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymers. While polymers such as ethylene-vinyl acetate and lactic acidglycolic acid enable sustained release of, certain hydrogels release ns for shorter time s. When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to re at 370 C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through isulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using riate additives, and developing specific polymer matrix compositions.

While the skilled artisan will understand that the dosage of each scIL-10 protein (including fusions of scIL-10 to an appropriate fusion partner and dimerized complexes thereof) in accordance with the invention will be dependent on the patient’s particular circumstances. The dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to mg/kg, of the host body weight. For example, dosages can be 0.3 mg/kg body weight, 1 mg/kg body , 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-30 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 . Dosage regimens include 1 mg/kg body weight or 3 mg/kg body weight by enous administration, with the protein being given using one of the following dosing schedules: every four weeks for six dosages, then every three months; every three weeks; 3 mg/kg body weight once ed by 1 mg/kg body weight every three weeks. ably, 0 fusion proteins (including fusions of each to an appropriate fusion partner and dimerized xes f in accordance with the invention is usually administered on multiple occasions. Intervals between single dosages can INCORPORATED BY REFERENCE (RULE 20.6) be, for example, weekly, y, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of the soluble n in the t. In some methods, dosage is adjusted to achieve a plasma concentration of soluble protein of about 0.1-1000 pg/ml and in some methods about 5- 300 mg/ml.

For therapeutic applications, scIL-IO proteins (including s of scIL-IO to an appropriate fusion r and dimerized complexes thereof) in accordance with the invention are administered to a subject, in a pharmaceutically acceptable dosage form. They can be administered intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, intra-ocular, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The n may also be administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by those of skill in the art as the clinical situation warrants.

Examples of le carriers, diluents and/or excipients include: (I) co's phosphate I5 buffered saline, pH about 7.4, containing about 1 mg/n11 to 25 mg/ml human serum n, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The methods of the present invention can be practiced in vitro, in vivo, or ex vivo. stration of scIL-10 proteins (including fusions of scIL-IO to an riate fusion partner and zed complexes thereof), and one or more additional therapeutic agents, whether co-administered or administered sequentially, may occur as described above for therapeutic ations. Suitable pharmaceutically able carriers, diluents, and excipients for co-administration will be understood by the skilled artisan to depend on the identity of the particular therapeutic agent being co-administered.

When present in an aqueous dosage form, rather than being lyophilized, scIL-lO (including fusions of scIL-IO to an appropriate fusion partner and dimerized complexes thereof) typically will be formulated at a concentration of about 0.1 mg/ml to 100 mg/ml, although wide ion outside of these ranges is permitted. For the treatment of disease, the appropriate dosage of scIL-10 (including fusions of scIL-IO to an appropriate fusion partner and zed xes thereof) will depend on the type of disease to be treated, the severity and course of the disease, whether the scIL-IO proteins (including fusions of scIL-10 to an appropriate fusion partner and dimerized complexes thereof) are administered for preventive or therapeutic es, the course of previous therapy, the patient's clinical history and response to the scIL-lO protein (including fusions of scIL-lO INCORPORATED BY REFERENCE (RULE 20.6) to an appropriate fusion partner and dimerized complexes thereof), and the discretion of the attending physician. The scIL-lO protein is suitably administered to the patient at one time or over a series of treatments.

EXAMPLES Example I: unsubstituted scIL-10 Desi u 0 scIL-10:CL:CHI:FC and scIL-10:CHI:CL:FC The O single chain fusion body molecule contains a covalently linked IL-lO homodimer fusion protein linked to the CL-CHl-Fc (Formula 3) domain or the CHl-CL-Fc of the IgGl heavy chain l and 2). The amino acid sequences of each molecule synthesized is found in Table 1.

Table 1 Protein Sequence tituted LLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFL scIL-10 (1021a PCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGGGSGGSPGQGTQS linker):CL:CH1: FPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLE EVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKA Fc MSEFDIFINYIEAYMTMKIRNGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN GGSGGGGSGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV :MHEALHNHYTQKSLSLSPGK (SEQ ID No- 23)fiSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNL Unsubstituted ELKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFL scIL-10 (10aa PCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGGGSGGSPGQGTQS linker):CHl :CL: ENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLE EVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKA Fc MSEFDIFINYIEAYMTMKIRNGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSG GGGSGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSGGEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV rscs ’MHEALH HYT KSLSLSPGK SEQ ID No- 24 Unsubstituted iSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNL LLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFL scIL-10 (Saa PCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGSPGQGTQSENSCTH linker):Fc FPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA ENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFI (Control) NYIEAYMTMKIRNEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG AL 5) Ex ression 0 scIL-10:CL:CH1:FC and scIL-10:CHI:CL:FC The genes were synthetically sized and ed in pcDNA3.l expression vector (GeneArt), and ently expressed in HEK293 cells using the 3 expression INCORPORATED BY REFERENCE (RULE 20.6) system (Life Technologies). Proteins were purified using Protein A (GE Healthcare) with low pH n and ed against 2L 1X PBS 2 times.

The molecules were analyzed by SDS PAGE gel under reducing and non-reducing conditions (. For ng and non-reducing conditions, 2.5ug of n was loaded onto an Any kD gel (Invitrogen) with a Precision Plus Protein Kaleidoscope standard (Invitrogen) (MW range IOkD — 250 kD). The molecule was characterized by analytical gel filtration on an XBridge Protein BEH SEC column, 200A, 3.5 um, 7.8 mm X 150 mm (Waters). The column was equilibrated and run at 0.9 ml/min with 100mM sodium phosphate pH 7.0 as a running buffer for all analyses. Purified samples (0.5mg/ml) were injected (15111) and eluted with a run time of 15 min (FIGs. 4 and 5).

Bioactivi 0 scIL-10:CL:CH1:FC and scIL-10:CH1:CL:FC In vitro bioactivity was assessed by evaluating the ability of scIL-10:CL:CH1:Fc and scIL-10:CH1:CL:Fc to stimulate proliferation of the mouse mast cell line MC/9 (ATCC CRL- 8306). The scIL-lO direct Fc fusion protein (scIL-10ch) was used as a l. For the assay, MC/9 cells were plated at 10,000 cells/well in DMEM media containing 10% heat inactivated fetal bovine serum, 2 mM glutamine and 0.05 mM 2-mercaptoethanol. Cells were incubated for 72 hours at 37°C, 5% CO2 with varying concentrations of human IL-10 (R&D Systems), scIL-10:CL:CH1:Fc, scIL-10:CH1:CL:Fc or scIL-10:Fc. After 72 hours, the cells were stained with CellTiter-Blue (Promega) for 4 hours at 37°C, 5% C02 ing to the manufacturer’s protocol. Fluorescent measurements were taken at 560/590 nm. IL-10 (ECso = 75 pM), scIL-102CL2CH12Fc (EC50 = 79 pM), scIL-102CH1:CL:Fc (EC50 = 93 pM) and scIL- :Fc (EC50 = 493 pM) were active in a dose dependent fashion (.

Mouse PK 0 0:C :C I:Fc and scIL-10:C 1:C :Fc scIL-10:CL:CH1:Fc, scIL-10:CH1:CL:Fc, and scIL-10:Fc pharrnacokinetics in mice were evaluated at a single intravenous doses of 0.5 mg/kg administered into tail vein and a single subcutaneous doses of 2.5 mg/kg administered into the interscapular . Blood samples (n=3 samples/time point/fusion protein) were collected at 0.083, 0.5 14 6 7 7 7 7 24,48, 96, 168, 192 and 216 hours after administration of scIL-10:CL:CH1:Fc, scIL-10:CH1:CL:Fc and scIL-10:Fc. For each time point/ fusion protein/route of administration, serum was pooled and concentrations were measured using rd MSD techniques. Bioanalytical data was subjected to non-compartmental cokinetic analysis using x WinNonlin 6.4 software. The pharmacokinetic parameter included standard pharmacokinetic parameters of INCORPORATED BY REFERENCE (RULE 20.6) maximum concentration lCmaxL time to maximum concentration {Tmax}: area under the time versus concentration curve (AUC), mean residence time (MRT), elimination half-life (tl/2)z clearance (CL), distribution volume at steady state (Vss), and bioavailability (%F) were ined and reported in Tables 2 and 3.

Table2 Compound Dose Dose ROA Cmax Tmax Cmax/D AUClast (~nMole/kg) (nM) (h) (nM/D) 0zCLzCnlec 140 scIL—10:CL:CH1:Fc scIL-10:CH1:CL:Fc scIL-10zCH1:CL:Fc (mL/hrlkg) 1. 811 59.57 1. 001 INCORPORATED BY REFERENCE (RULE 20.6) Example 2: 10)-L1-HINGE-Fc Fusion Proteins.

Design of scIL-lO variant fusion proteins.

The scIL-IO of Formula 1 are fused to a single chain Fc linker of Formula 2 wherein L1 is CL-CHl-Fc as per Formula 3. The amino acid sequences of each full length scIL Ll-HINGE-Fc fusion variant protein synthesized is found in Table 4. The ption column of Table 4 indicates the scIL-lO used in the construct with the fusion partner. For example thL-10zlinkerzD41F tes that in accordance with Formula 1, the first monomer subunit is wt IL-10 of SEQ ID NO: 1 and is therefore unsubstituted linked to a linker which is in turn linked to the second monomer subunit wherein the wtle of SEQ ID NO: I is substituted at amino acid 41 such that the cine at amino acid 41 is substituted with phenylalanine (D4 1 F).

For expression in mammalian cells, the N-terminal leader sequence of SEQ ID NO: 48 was added to each of the protein sequences found in Table 4.

Table 4 Description Amino Acid Sequence/SEQ ID NO SCIL-10:CL:CH1:FC SPGQGTQs3N SCTIFPGNLP VWLRDLRDAF SRVKTFFQWK DQDDVDDDKR 5.33DFKGYD GCQAT.SEMIQ FYT.ERVMPQA 3NQDPD:KAH VVSLG3NLKT LRLRLRRCHR FLPC3VKSKA V3QVKNAFVK LQ3KGIYKAM SLFDIFINYI 3AYMTMKIRN GGSGGGGSGG SPGQGTQS3N SCTHFPGNLP NWLRDLRDAF SRVKTFFQMK DQLD LLLKE SLLEDFKGYL GCQAT.S MIQ rY333VMPQA 3VQDPDIKAH V'SLGENLKT LRLRLRRCIR FLPC3N<SKA V3QV<VAFNK LQ3KGIYKAM SthItINYI KIRN GGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN AKVQ WKVDVALQSG NSQ3SV33QD S<JSTYSLSS TLTLS<ADY3 <3KVYAC3VT HQGLSS?V"K CGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAP SSKS"SGG"A ALGCLV<DYF SWNS VHTF SGLY TVPS SSLGTQmYIC NVVHKPSN"K VDKRV3PKsc D<THTCPPCP APEL3GGPSV FLFPP.<P3D3 LMISRTPEVT CVVVDVSHED WYVD GVEVHNAK"K PREEQY STY RVVSVLTVLH KEYK C(VSNKALPA PIEK"ISKAK GQPRZPQVY" DPPSR33M3K NQVSLTCLVK GFYPSDIAV3 WESNGQ?ENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALINHYTQKS < (s3Q I) 30: 12) M22A:Iinker:D41N SPGQGTQSEN SCTHFPGNLP NALRD3RDAF (R1+R2 mutant) SRVKTF3QMK DQT.DNT.LLK3 STE3DF<GYT. GCQAT.S3MIQ FY133VMPQA 3NQDPDIKAH VNSLG3 LKT LRLRLRRCHR FLPCENKSKA V3QVKVAFNK YKAM INYI EAYWTWKIRN GGSGGGGSGG SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF SRV<TFFQMK NQT.D rrrK3 SDD3DEKGYD GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT LRLR3RRCHR FLPCENKSKA VEQVKNAFNK LQE<GIYKAM SEFDIFINYI EAYW“WKIRN GGGGSGGGGS RTVAAPSVFI FPPSD3QLKS GTASVVCLLV NFYPR3AKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLss TLTLSKADYE K3KVYACEVT HQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAP SS<STSGG”A ALGCLVKDYF SWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSVTK VDKRVEPKSC PPCP APELLGGPSV FLFPPKPKDT LWISRTPEVT CVVVDVSH3D P3VKrNWYVD INCORPORATED BY REFERENCE (RULE 20.6) A<TK PR4 *QYNSTY RVVSVLTVLH QDWLNGKLYK ALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE W‘SNGQ?*VN YK"TPPVLDS DGSFFLYSKL WQQG NVFSCSVMH.L ALiNHYTQ<S LSLSPGK (S 3Q ID NO: 44) D41N:|inker:M22A SPGQGTQSLN ScfiiFPGNLP VMLRDLRDAF (R1+R2rnutann FQMK NQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA EVQDPDIKAH NLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFVK LQLKGIYKAM S.LtDItINYI .LAYMTMKIRN GGSGGGGSGG QSEN SCTHFPGNLP N SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQALSEMIQ ENQDPDIKAH VZSLGENLKT LRLRLRRCflR FLPCENKSKA LQLKGIYKAM S.LEDIEINYI .LAYMTMKIRN GGGGSGGGGS FPPSDEQLKS CLLV VFYPREAKVQ WKVDVALQSG SLSS TLTLS<ADYE {{KVYACLVT HQGLSS?V”K SFNRGLCGGG GSGGGGSGGG GSGGGGSAST PLAP SS_<STSGGTA ALGCLVKJYF PEPVTVSJNS GALTSGVHTF SGLY SLSSVVTVPS SSLGTQmYIC NVVHKPSV"K VDKRVEPKSC D (THTCPPCP APELLGGPSV FLFPP<P<Dr1 LMISRTPEVT CVVVDVSH*D P*VKENWYVD GVEVHNAKHK PREEQY‘STY RVVSVLTVLH QDWLNGKEYK C(VSNKALPA PILKTISiAK GQPRLPQVYr1 T.PPSR*HLW”K NQVSLTCLVK GFYPSDIAVL WLSNGQPLVN YKmTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHY"Q<S LSLSPG< (SEQ ID V0: 43) SPGQGTQSEN SC"{FPGNLP NWLRDLRJAF thL-10:|inker:M22A, SRVKTFFQWK DQEDVKT.TK* SWE‘DEKGYE GCQATS MIQ tYl4‘VMPQA D41N (R1+R2 mutant) 3 _KAH 3 LKT LRLRLRRCHR FLPCLV<SKA VEQV<NAFNK SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSEN SCTHFPGNLP NALRDLRDAF SRV<TFFQMK NQLD LRLKL SRLLDEKGYR GCQALS*MIQ MPQA EVQDPDIKAH VNSLGEVLK" RCHR FLPCENKSKA VEQVKNAFNK LQEKGIYKAM SEFDIFIVYI EAYWTWKIRN GGGGSGGGGS RTVAAPSVFI FPPSDLQLKS GTASVVCLLV NFYP?.LA<VQ WKVDNALQSG TLQD S<DSTYSLSS TLTLS<A>Y3 KHKVYAC.LVT HQGLSS?V"K SFNRGECGGG GSGGGGSGGG GSGGGGSAS" KGPSVFPLAP SS<S"SGG"A ALGCLV<DYF PLPVTVSWNS GALTSGViTF PAVLQSSGLY TVPS SSLGTQTYIC SNTK VDKRVEPKSC PPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSAED PEVKFNWYVJ GVEVHNA<"K PRLLQY STY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEK"ISKAK GQPREPQVY" LPPSREEMTK NQVSLTCLVK GFYPSDIAVE W‘SNGQ?*VN YK"TPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALiNHYTQ<S < (s 3Q I) V0: 42) WHLJOJmkenD41N (R1 SPGQGTQSLN GNLP VMLRDLRDAF mutant) SRVKTFFQWK DQT.DNRT.LK4 STTLDEKGYR GCQARSLMIQ rYtaaVMPQA EVQDPDIKAH VVSLGE LKT LRLRLRRCHR FLPCEN<SKA VEQV<VAFVK LQLKGIYKAM S.LtDItINYI LAYWTWKIRN GGSGGGGSGG SPGQGTQSEN GNLP NWLQDLQDAF SRVKTFFQMK NQLDNLLLKE SLLEDFKGYL GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VZSLGENLKT LRLRLRRCflR FLPCENKSKA V.LQVKVAENK LQLI]. (GIYKAM SLEDIEINYI .AYM'WIKI RN GGGGSGGGGS RTVAAPSVFI FPPSDLQLKS GTASVVCLLV VFYPRLAKVQ WKVDVALQSG NSQ‘SVr1 QD SKJSTYSLSS TLTLSKADY.3 {{KVYACEVT HQGLSS?V"K SFNRGLCGGG GSGGGGSGGG GSGGGGSAST {GPSVFPLAP SS_<STSGGTA ALGCLVKJYF PEPVTVSJNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQfiYIC NVVHKPSV"K VDKRVEPKSC D PCP APELLGGPSV FLFPP<P<Dr1 LMISRTPEVT CVVVDVSH*D P*VKENWYVD GVEVHNAKHK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK ALPA PILKTISiAK QVYr1 T.PPSR*HLW”K NQVSLTCLVK IAVL WLSNGQ?*VN VLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHY"QKS LSLSPGK (SEQ ID V0: 41) M22AflMkenD41A SPGQGTQSEN SC"{FPGNLP NALRDLRJAF (R1+R2rnutann FQWK DQEDVTmTTK* SWE‘DEKGYE GCQATS MIQ tYl4‘VMPQA 3 _KAH VNSLGLNLKT LRLRLRRCHR FLPCLV<SKA VEQV<NAFNK SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF SRV<TFFQMK AQtD LRLKL SRLLDEKGYR GCQALS*MIQ MPQA ENQDPDIKAH VNSLGEVLKT LRLRLRRCHR INCORPORATEDITKREFERENCE(RULE206) 2017/038747 KSKA VLQVKVAENK LQLKGIYKAM S.LEDIEINYI LAYMTMKIRN GGGGSGGGGS RTVAAPSVEI EPPSDEQLKS GTASVVCLLV VEYPREAKVQ WKVDVALQSG NSQ‘SV"*QD SKJSTYSLSS TLTLS<ADYE {{KVYACEVT HQGLSS?V"K SENRGECGGG GSGGGGSGGG GSGGGGSAST {GPSVEPLAP SS_<STSGGTA ALGCLVKJYE PEPVTVSJNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQ"YIC NVVHKPSV"K VDKRVEPKSC D (THTCPPCP GPSV ELEPPKPKDr1 LMISRTPEVT CVVVDVSH*D P*VKPNWYVD GVEVHNAKHK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTIS<AK GQPREPQVYr1 TPPSRLLW”K NQVSLTCLVK GEYPSDIAVE WLSNGQ94NN YKflTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHY"QKS K (SEQ ID V0: 40) SPGQGTQSEN SC"HFPGNLP NWLRDLRJAE D41A:anenhfl22A SRVKTFFQMK mTTK* SWE‘DP<GYE GCQATS MIQ tYl4‘VMPQA : :KAH VNSLGENLKT LRLRLRRCHR ELPCEV<SKA (R1+R2 ) VEQV<NAENK SEFDIFINYI EAYMTMKIRN GGSGGGGSGG QSEN SCTHFPGNLP NALRDLRDAF SRV<TFFQMK DQT.D LT.T.K* SRLLDEKGYR *MIQ tYT.* VMPQA EVQDPDIKAH VNSLGEVLKr1 LRLRLRRCHR KSKA VEQV<NAFNK LQEKGIYKAM SEEDIEIVY: EAYWTWKIRN GGGGSGGGGS RTVAAPSVFI EPPSDEQLKS GTASVVCLLV NEYP?.3A<VQ WKVDNALQSG NSQLSV"4QD S<DSTYSLSS TLTLS{A)Y.3 {KVYAC.LVT HQGLSS?V"K SFNRGECGGG GSGGGGSGGG GSGGGGSASr1 GPSVEPLAP SS<STSGG"A ALGCLV<DYF PEPVTVSWNS GALTSGVTTE PAVLQSSGLY SLSSVVTVPS SSLGTQmYIC SV"K VDKRVEP<SC D<THTCPPCP APELLGGPSV FLFPPKP<D" LWISRTPLVT CVVVDVSTLD P LVKENWYVD A<mK PRLLQY STY RVVSVLTVLH QDWLNGKEYK C (VSNKALPA PIEKTISKAK GQPREPQVY" LPPSREEW"K NQVSLTCLVK GEYPSDIAVE W‘SNGQP*VN YK"TPPVLDS DGSEELYSKL TVDKSRWQQG NVESCSVMHE ALiNHYTQ<S LSLSPG< (s 3Q ID NO: 39) thL-10:|inker:M22A, SPGQGTQSEN SC"{FPGNLP VMLRDLRQAE D41A (R1+R2 mutant) SRVKTFFQWK DQTDNTRTK4 STPLDEKGYR GCQARSLMIQ PY144VMPQA EVQDPDIKAH VVSLGE LKT LRLRLRRCHR ELPCEN<SKA VEQV<NAEVK LQEKGIYKAM S.LtDItINYI LAYWTMKIRN GGSGGGGSGG SPGQGTQSEN SCTHEPGNLP NAL?DL?DAE SRV<TEEQMK LLKE SLLEDEKGYL GCQALSEMIQ MPQA ENQDPDIKAH VZSLGENLKT LRLRLRRCflR KSKA V.LQVKVAENK LQ_LI]. (GIYKAM INYI .LAYMTTMKI RN GGGGSGGGGS RTVAAPSVEI EPPSDEQLKS GTASVVCLLV VEYPREAKVQ WKVDVALQSG NSQ‘SVr1 QD SKJSTYSLSS TLTLSKADYE {{KVYACEVT HQGLSSPV"K SENRGECGGG GSGGGGSGGG GSGGGGSAST {GPSVEPLAP SS_<STSGGTA ALGCLVKJYE PEPVTVSJNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS "YIC SV"K VDKRVEPKSC D (THTCPPCP APELLGGPSV PKDr1 LMISRTPEVT CVVVDVSH*D P*VKPNWYVD GVEVHNAKHK PREEQYNSTY RVVSVLTVLH KEYK C(VSNKALPA PIEKTISiAK GQPRLPQVYr1 TPPSR‘H‘WfiK NQVSLTCLVK GEYPSDIAVE W4SNGQ?*VN YKmTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHY"QKS LSLSPGK (SEQ ID V0: 38) wHLJDflmkenD41A (R1 PGQGTQSEN SC"{FPGNLP RJAE mutant) DQEDVTHTTK* S.R*DEKGYE GCQARS‘MIQ tYl4‘VMPQA VNSLGLNLKT LRLRLRRCHR ELPCEV<SKA VEQV<NAENK SEFDIFINYI GGSGGGGSGG SPGQGTQSEN SCTHEPGNLP NMLRDLRDAE AQLD LLLKE SLLEDEKGYL GCQALS*MIQ LRLRLRRCHR tYR**VMPQA 4IO u "U u H EI VNSLGIZVLKr1; ELPCENKSKA mmnjb'mm VEQVKNAENK to m A G) Hi SEEDIEIVY: EAYMTWKIRN GGGGSGGGGS LKS GTASVVCLLV NEYP?.3A<VQ WKVDNALQSG {DSTYSLSS TLTLS<ADYE KHKVYACLVT HQGLSS?V"K GGG GSGGGGSASr1 KGPSVEPLAP GG"A ALGCLVKDYF IT'ZtU LPVTVSWNS GALTSGVTTE PAVLQSSGLY SLSSVVTVPS SSLGTQmYIC VNHKPSN"K VDKRVEP<SC DKTHTCPPCP APELLGGPSV PKDT MISRTPEVT CVVVDVSJED PEVKFNWYVD GVEVHNA<mK PRLLQYNSTY RVVSVLTVLH QDWLNGKLYK CKVSNKALPA PIEK"ISKAK GQPREPQVY" LPPSREEM"K NQVSLTCLVK IAVE W‘SNGQ?*NN VLDS DGSEFLYSKL TVDKSRWQQG NVESCSVMHE INCORPORATEDITKREFERENCE(RULE206) ALHNHYTQ<S LSLSPGK (SEQ ID NO: 37 ) M22A:linker:D41F QSEN SCTHFPGNLP VALRDLRDAF (R1+R2 ) SRVKTFFQWK DQtDNtttK 3 STI*DEKGYR GCQARS*MIQ tYL44VMPQA ENQDPDIKAH NLKT LRLQLRRCHR FLPCENKSKA VEQVKNAFWK YKAM jEDIEINYI LAYWTMKIRN GGSGGGGSGG QSEN SCTHFPGNLP SRV.<TFFQMK FQLDNL3LKE S3LEJFKGYL GCQALSEMIQ ENQDPDIKAH < SLGEVLKT LRLRLRRCHR FLPCENKSKA 3<GIYKAM m LEDIEINYI L*AYMTMKIRN GGGGSGGGGS FPPSDEQLKS O AKVQ WKVDNALQSG SKJSTYSLSS Fof!» U) U ._<m aflKVYACEVT HQGLSSPV"K GSGGGGSGGG G AGPSVFPLAP SSKSTSGG"A PEPVTVSJNS m:1) L—'3 U2 C)<{Ia ”j GLY SLSSVVTVPS SSLGTQ"YIC VVVHKPSV"K < u m /UflLJ "U 71 m 03 FLFPP<PKD" LMISRmPEVT OWUWKVSNKALPA{THTCPPCP APELLGGPSV fiVKENWYV) GVEVHNAKTK PREEQYNSTY RVVSV3TV3H PI3KTIS<AK GQPREPQVY" RPPSR *W”K NQVSLTCLVK GFYPSDIAV _‘ 3 4 W4SNGQ?*NN YKTTPPVLDS DGSFFLYSKL TVD<SRWQQG NVFSCSVMH_‘ 4 ALHNHYHQKS LSLSPGK (SEQ ID V0: ) D41F:anenhfl22A SPGQGTQSEN SCTHFPGNLP VWLRDL?3AF (R1+R2 mutant) S_RVKTFFQMK GCQA3SEMIQ FYLEEVMPQA 3 :KAH VVSLGENLKT LRLRLRRCHR FLPCEVKSKA <EQVKVAFVK DIEINYI GGSGGGGSGG w PGQGTQSEN SCTHFPGNLP NALRDLRDAF DQT.D T.T.T.K* m 4! 4. *DtKGYK GCQAtS3MIQ fifiVMPQA F VNSLGENLKT b 3L3L2RC3R FLPCENKSKA VEQVKNAFNK SEFDIFINYI mAYMTMKIRN GGGGSGGGGS FPPSDEQLKS GTASVVCLLV 2 “j..< "U Q WKVDNALQSG *QD S<DSTYSLSS ADY3 W {KVYACEVT HQGLSS?V”K SFN?GECGGG GSGGGGSGGG SAST NGPSVFPLAP GG”A ALGCLV<DYF PEPVTVSWNS GALTSGVITF AVLQSSGLY SLSSVVTVPS SSLGTQHYIC VVNHKPSNTK VDKRVEP<SC {THTCPPCP APELLGGPSV FLFPPKP<DT LWISRTPEVT CVVVDVSHED EVKFNWYVD GVEVHNAK"K PR**QY STY RVVSVLTVLH QDWLNGKEYK {VSNKALPA PIEKTISKAK QVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVADS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALiNHYTQ<S LSLSPG< (SEQ ID NO: 28 ) SPGQGTQSEN SC"{FPGNLP VWLRDLRDAF thL-10:|inker:M22A, SRVKTFFQWK DQT.DNIT.IK* STI*DEKGYR GCQARS*MIQ tYL**VMPQA D41F (R1+R2 mutant) ENQDPDIKAH VVSLGE‘LKT LQLQLRRCHR FLPCENKSKA VEQVKNAFWK LQ3KGIYKAM S.LEDIE'INYI jAYWTMKIRN GGSGGGGSGG SPGQGTQSEN GNLP NALRDLRDAF SRV<TFFQMK FQLDNLLLK4 S1143hKGYR GCQALSEMIQ MPQA EVQDPDIKAH < SLGEVLKT LRLRLRRCHR FLPCENKSKA VLQVKVAtNK LQL<GIYKAM m LEDIEINYI L*AYWTMKIRN GGGGSGGGGS RTVAAPSVFI FPPSDEQLKS O 4FYPQEAKVQ WKVDNALQSG NSQESVTEQD SKJSTYSLSS F U ._<m aflKVYACEVT HQGLSSPV"K SFNRGECGGG GSGGGGSGGG G (UL—33> U) AGPSVFPLAP SSKSTSGG"A ALGCLVKDYF PEPVTVSJNS m:1) L—'3 U2 C)<:1:a “j AVLQSSGLY TVPS "YIC SV"K < u m /UflLJ "U 71 m 03 APELLGGPSV FLFPP<PKD" LWISRTPEVT fiVKENWYV) PREEQYNSTY RVVSVLTV3H OWUFU KVSNKALPA{THTCPPCP AKTK PIEKTIS<AK GQPREPQVY" LPPSREEW"K GFYPSDIAVE W4SNGQ?*NN VLDS DGSFFLYSKL TVD<SRWQQG NVFSCSVMHE3 ALHNHYHQKS LSLSPGK (SEQ ID NO: 27 ) SPGQGTQSEN SCTHFPGNLP VALRDLRDAF kenD41F S_RVKTFFQMK FQLDN3LLKE SLLEDFKGYL GCQA3SEMIQ FYLEEVMPQA 3 :KAH VVSLGENLKT LRLRLRRCHR KSKA < (R1+R2 mutant) EQVKNAFVK S.3tDItINYI LAYMTMKIRN GGSGGGGSGG w PGQGTQSEN SCTHFPGNLP NWL?3LRDAF SRVKTFFQMK DQLD‘LLLKE m LLEDFKGYL fiMIQ fifiVMPQA ENQDPDIKAH NLKT b RLRLRRCIR FLPCENKSKA VEQVKNAFNK 3QEKGIYKAM SEFDIFINYI mAYMTMKIRN GGGGSGGGGS RTVAAPSVFI QLKS GTASVVCLLV 2FYPREA<VQ WKVDNALQSG NSQ*SVT* QD SKDSTYSLSS TLTLS<ADY3 W {KVYACEVT INCORPORATEDITKREFERENCE(RULE206) HQGLSSPV"K SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAP SSKSTSGG"A ALGCLVKDYF PEPVTVSJNS GALTSGVHTF PAVLQSSGLY TVPS TYIC NVNHKPSV"K VDKRV.3PKSC D<THTCPPCP APELLGGPSV FLFPP<PKDr1 LMISRflPEVT CVVVDVSH*'D PfiVKPNWYV) GVEVHNAKTK PREEQYNSTY TV3H QDWLNGKEYK CKVSNKALPA S<AK GQPREPQVY" T.PPSR 3 *W"K NQVSLTCLVK GFYPSDIAV _‘43 W*SNGQP*NN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMH_‘ 4 ALHNHYHQKS LSLSPGK (SEQ ID V0: 26) MZZAJMkenMZZA (R2 SPGQGTQSEN SCTHFPGNLP NALRDL?3AF mutant) SRVKTFFQMK DQLDN3LLKE SLLEDF_<GYL GCQA3SEMIQ FYLEEVMPQA D.KAH VNSLGENLKT LRLRLRRCHR FLPCEVKSKA AFNK LQEKGIYKAM SEFDIFINYI EAYMTWKIRN GGSGGGGSGG m PGQGTQSEN SCTHFPGNLP NALRDLRDAF SRVKTFFQMK DQT.D T.T.T.K* m 3. 4. *DtKGYP GCQAISfiMIQ EYPfifiVMPQA EVQDPDIKAH VNSLGENLKT b RLRLRRCHR FLPCENKSKA VEQVKNAFNK 3QEKGIYKAM SEFDIFINYI mAYMTMKIRN GGGGSGGGGS SVFI FPPSDEQLKS GTASVVCLLV 2 “j..< "U 3U3A<VQ WKVDNALQSG NSQ*SV"*QD S<DSTYSLSS TLTLS<ADYE W {KVYACEVT HQGLSS?V”K SFN?GECGGG GSGGGGSGGG GSGGGGSAST NGPSVFPLAP SS<STSGG”A ALGCLV<DYF PEPVTVSWNS GALTSGVITF OrUUrUAVLQSSGLY SLSSVVTVPS SSLGTQmYIC VVNHKPSN”K VDKRVEP<SC (THTCPPCP GPSV FLFPPKP<Dr1 LWISRTPEVT CVVVDVSHED EVKFNWYVD GVEVHNA<"K PR4 QY STY RVVSVLTVLH QDWLNGKEYK {VSNKALPA PI3KTIS<AK GQPREPQVYr1 TPPSRfl*'M"K NQVSLTCLVK GFYPSDIAVE W3SNGQ33VN YKfiTPPVLDS )GSFFLYSKL TVDKSRWQQG VMH3 ALINHYTQ<S LSLSPG< (S 3Q I 3 30: 25) thL-10:|inker:M22A (R2 SPGQGTQSEN SC"{FPGNLP VMLRDLRDAF mutant) SRVKTFFQWK DQT.DNPT.IK* STI*DE<GYR GCQAPS*MIQ EYE3 *VMPQA ENQDPDIKAH VVSLGE‘LKT LRLQLQQCHR KSKA VEQVKNAFVK LQ3KGIYKAM S.3EDIP'INYI 3AYWTW<IRN GGSGGGGSGG SPGQGTQSEN SCTHFPGNLP NALRDLRDAF SRV<TFFQMK DQLDNLRLK4 S1143hKGYP GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH < SLGEVLKT RCHR FLPCENKSKA V3QVKVAPNK IYKAM m 3tDItINYI L3AYWTMKIRN GGGS RTVAAPSVFI FPPSDEQLKS OTASVVCLLV 4FYPQEAKVQ WKVDNALQSG NSQESVTEQD SKJSTYSLSS Fa L—' [/2 XADYE aflKVYACEVT PV"K SFNRGECGGG3' GSGGGGSGGG GSGGGGSAST AGPSVFPLAP SSKSTSGG"A ALGCLVKDYF SJNS mALTSGVHTF AVLQSSGLY SLSSVVTVPS SSLGTQ"YIC VVVHKPSV"K <DKRVEPKSC {THTCPPCP APELLGGPSV FLFPP<PKD" LMISRTPEVT OVVVDVSH“D GVEVHNAKTK RVVSVLTV3H QDWLNGKEYK OWUWKVSNKALPAfiVKPNWYV) PREEQYNSTY PIEKTIS<AK GQPREPQVY" LPPSREEW”K NQVSLTCLVK GFYPSDIAVE W+SNGQP+NN YKTTPPVLDS DGSFFLYSKL WQQG NVFSCSVMHE3 ALHNHYHQKS LSLSPGK (SEQ ID NO: 24) SPGQGTQSEN SCTHFPGNLP VALRDLRDAF M22A:Iinker:wtlL-10 (R2 FFQMK DQLDN3LLKE SLLEDFKGYL GCQA3SEMIQ FYLEEVMPQA 3 :KAH VVSLGENLKT LRLRLRRCHR FLPCEVKSKA < mutant) EQVKNAFVK S.3tDItINYI LAYMTMKIRN GGSGGGGSGG w PGQGTQSEN SCTHFPGNLP NWLRDLRDAF SRVKTFFQMK DQT.D T.T.T.K* m 3. 4. *DtKGYP GCQAISfiMIQ VMPQA EVQDPDIKAH VNSLGENLKT b RLRLRRCIR FLPCENKSKA VEQVKNAFNK 3QEKGIYKAM SEFDIFINYI mAYMTMKIRN GGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLV 2W._< "U 3UE <VQ WKVDNALQSG "*QD S<DSTYSLSS TLTLS<ADY3 W {KVYACEVT HQGLSS?V”K SFN?GECGGG GSGGGGSGGG SAST NGPSVFPLAP SS<STSGG"A ALGCLVKDYF SWNS GALTSGVITF AVLQSSGLY SLSSVVTVPS SSLGTQmYIC NVNHKPSN"K VDKRVEP<SC (THTCPPCP GPSV FLFPPKPKDr1 CVVVDVSHED GVEVHNAK"K PR‘ QYNSTY RVVSVLTVLH KEYK OrUUrU EVKFNWYVD{VSNKALPA PIEKTISKAK GQPR3PQVYr1 *'M"K NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS YSKL TVDKSRWQQG VMHE ALINHYTQKS LSLSPGK (S 3Q ID no: 23) INCORPORATEDITKREFERENCE(RULE206) SPGQGTQSEN SCTTFPGNLP VMLRDLRDAF D41FflnkenD41F (R1 SRVKTFFQMK FQT.TUTTKF STEFDFKGYT. GCQAT.SFMIQ FYLFPVMPQA ENQDPDZKAH VVSLGENLKT RCHR FLPGEVKSKA mutant) AFVK LQ3KGIYKAM IEINYI MKIRN GGSGGGGSGG SPGQGTQSEN SCTHFPGNLP RDAF FQMK FQLDN3LLKE S3LEDFKGYL GCQAT.S1MIQ E . ENQDPDIKAH V'SLGENLKT LRLRLRRC3R FLPC3N<SKA V.3QVKVAENK LQ3KGIYKAM IVYI L1.AYMTMKIRN GGGGSGGGGS FPPSDEQLKS CLLV '71 ._< I'U W9KVQ WKVDVALQSG NSQ3 SKDSTYSLSS TLTLS<A>Y3 PV"K SFNRGECGGG SGGG GSGGGGSAST AAA {KVYACEVTGPSVFPLAP SSKSTSGG"A ALGCLVKDYF PEPVTVSWNS GALTSGVHTF AVLQSSGLY SLSSVVTVPS SSLGTQ"YIC NVVHKPSN"K VDKRV3P<sc {THTCPPCP APELLGGPSV FLFPP<PKDT PEVT CVVVDVSHED PR3ZQYNSTY RVVSVLTVLH QDWLNGKEYK OrUUrU III<1 71 ”j2E: '_<<3 GVEVHNAK"K LPA PIEKTISKAK GQPR3PQVYT RPPSR11MmK NQVSLTCLVK GFYPSDIAV.3'3' WESNGQ?EVN YK"TPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALiNHYTQ<S LSLSPGK (s 3Q I) V0: 22) thL-10:|inkerD41F (R1 SPGQGTQSEN SC"{FPGNLP NMLRDLRDAF mutant) SRVKTFFQMK DQKDNT.RRK3 SWP3DE<GYE GCQAT.S1'MIQ t'Yl33VMPQA ENQDPDIKAH VVSLG.3NLKT LRLRLRRCHR FLPC3N<SKA VEQV<VAFNK LQEKGIYKAM SEFDIFINYI EAYMTM<IRN GGSGGGGSGG QSEN GNLP NMLRDLRDAF SRV<TFFQMK FQmD mmmK3 SER1DEKGYE GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGEVLKT FLPQ3NKSKA V3QVKVAENK LQ.3<GIYKAM S3t'DIt'IVYI 3 GGGGSGGGGS RTVAAPSVFI FPPSD.3QLKS GTASVVCLLV VFYPREAKVQ WKVDVALQSG NSQESV"EQD SKJSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSS?V"K SFNRGECGGG GSGGGGSGGG GSGGGGSAST {GPSVFPLAP SS<STSGG”A ALGCLV<DY3 PEPVTVSJNS GALTSGViTF PAVLQSSGLY TVPS SS3GTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV FLFPP<PKDT PEVT CVVVDVS{1D P1VKENWYVD GVEVHNAK"K PR11QY STY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE W3SNGQ33VN YK"TPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE TQ<S LSLSPG< (G 21) inker:wt|L-10 (R1 QSEN SC"{FPGNLP RJAF ) SRVKT33QWK FQT.DNmeK3 SiTFDFKGYL GCQARSFMIQ FYLFPVMPQA EVQDPDIKAH VVSLGE LKT LRLRLRRCHR FLPCENKSKA EQVKNAFVK LQ3KGIYKAM S3EDIEINYI 3AYMTMKIRN GGSGGGGSGG PGQGTQS3N SCTHFPGNLP NMLRD3RDAF SRVKTFFQMK DQLDNLLLKE 3LEDFKGYL GCQALSEMIQ 3YLEEVMPQA IKAH V'SLGENLKT L—'(/2(/}< RLRLRRCHR FLPCENKSKA V3QVKVAENK LQ3KGIYKAM S.3tDItIVYI GGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLV r11:33<3< m2weg2:3 ,3 :UzKVQ WKVDVALQSG NSQfisvmfiQD SKDSTYSLSS TLTLSiADYE {KVYACEVT HQGLSSPV"K SFNRGECGGG GSGGGGSGGG GSGGGGSAST AAALLGPSVFPLAP SSKSTSGG"A ALGCLVKDYF PEPVTVSWNS GALTSGVHTF OrUUrU III<1 71 ”j2E: '_<<AVLQSSGLY SLSSVVTVPS SSLGTQ"YIC NVVHKPSN"K VDKRVEP<SC {THTCPPCP APELLGGPSV FLFPP<PKDT LMISRTPEVT CVVVDVSHED 3 GVEVHNA<”K PR3QYNSTY TVLH KEYK {VSNKALPA PI3KTIS<AK GQPR3PQVYT T.PPSRA. 1M"K NQVSLTCLVK GFYPSDIAV W1SNGQP1NN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMH_‘ 3 ALiNHY"QKS LSLSPGK (s 3Q I) V0: 20) thL-10:|inker:l87A SPGQGTQSEN SCTiFPGNLP NMLQDLQDAF (vlL10 mutant) SRVKTFFQMK DQRDVTMTTK1 SWP3DE<GYE GCQAT.S1'MIQ VMPQA 3 _KAH VNSLG3NLKT LRLRLRRCHR FLPCEV<SKA VEQV<VAFNK SEFDIFINYI EAYMTMKIRN GGSGGGGSGG SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF SRV<TFFQMK DQmD mmmK3 SER1DEKGYE GCQARS3MIQ t'YE3'VMPQA ENQDPDAKAH VNSLGEVLKr1 LRLRLRRCHR FLPCENKSKA VEQVKNAFNK 3QEKGIYKAM SEFDIFINY: EAYMTMKIRN GGGGSGGGGS RTVAAPSVFI FPPSDEQLKS CLLV NFYPR3AKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSASr1 KGPSVFPLAP INCORPORATEDITYREFERENCE(RULE206) SS<STSGGTA KDYF PEPVTVSWNS GALTSGVITF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC VVNHKPSVTK VDKRVEPKSC DKTHTCPPCP GPSV FLFPPKPKDT PEVT CVVVDVS{*D P‘VKtNWYVD GVEVHNA<TK PR‘11QYNSTY RVVSVLTVLH QDWLNGKEYK C(VSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE PLVN VLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEA.

ALTNHYTQKS LSLSPGK (SEQ ID NO: l9) SPGQGTQSEN SCTTFPGNLP VMLRDLRJAF |87A:|inker:wt|L-10 SEVKTFEQWK DQKDVELTKR SWTEDFKGYE EMIQ FYtERVMPQA VNSLGENLKT LRLRLRRCHR (vlL10 mutant) ENQDPDAKAH VEQVKNAFNK LQEKGIYKAM SLEDIEINYI LAYMTMKIRN SPGQGTQSEN SCTHFPGNLP NWLRDLRDAF SRVKTFFQMK SLLEDFKGYL GCQATS MIQ t “*VMPQA ENQDPDIKAH LRLRLRRCTR FLPC.EN<SKA VLQV<VAENK LQLKGIYKAM L“AYMTMKIRN GGGGSGGGGS RTVAAPSVFI FPPSDEQLKS ZFYPREAKVQ WKVDVALQSG NSQLSVTLQD SKJSTYSLSS TLTLS<ADYE A EVT HQGLSS?VTK SFNRGECGGG GSGGGGSGGGJ. GSGGGGSAST AGPSVFPLAP SS<STSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF GLY SLSSVVTVPS SSLGTQTYIC NVVHKPSVTK VDKRVEPKSC {THTCPPCP APELLGGPSV FLFPP<P<DT PEVT CVVVDVSH‘D QDWLNGKEYK OWUW {VSNKALPA*VKENWYV) GVEVHNA<TK PREEQY STY RVVSVLTVLH PIEKTIS<AK GQPREPQVYr1 EPPSR44WTK NQVSLTCLVK GFYPSDIAVE WfiSNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALINHYTQKS LSLSPG< (SEQ ID V0: 18) |87A:|inker:l87A (vlL10 SPGQGTQSEN GNLP NMLRDLRJAF mutant) SRVKTFEQMK DQTDVTTTKL S‘TLDE<GYT GCQARSLMIQ w 4. L *VMPQA .EVQDPDAKAH VNSLG.E LKT LRLRLRRCHR FLPCEV<SKA < {VAFNK LQEKGIYKAM INYI EAYWTW<IRN GGSGGGGSGG m FUNK (DIO<QGTQSEN SCTHFPGNLP NMLRDLRDAF SRV<TFFQMK DQRD RT.TKa m 4! 4. fiDEKGYT GCQALSLMIQ EYLL VMPQA EVQDPDAKAH VNSLGEVLKT HNF<k<w NLRRCHR FLPCEN<SKA VEQV<NAFNK YKAM SEFDIFIVYI mV STWKIRN GGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLV 2W W REAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE CEVT PVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAP SS<STSGGTA ALGCLVKDYF PEPVTVSTNS GALTSGVITF PAVLQSSGLY SLSSVVTVPS TYIC VVNHKPSVTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV PKDT LWISRTPEVT CVVVDVS{*D P‘VKtNWYVD GVEVHNAKTK PR‘1QYNSTY RVVSVLTVLH QDWLNGKEYK C(VSNKALPA PIEKTISKAK GQPREPQVYT EMTK NQVSLTCLVK GFYPSDIAVE WESNGQ?EVN YKTTPPVLDS DGSFFLYSKL WQQG NVFSCSVMHE TQKS LSLSPGK (SEQ ID NO: l7) MZZA SPGQGTQSEN SCTIFPGNLP VALRDLRIAF D41F:anenhfl22A, D41F SRVKTFEQWK FQEDVELEKR SWTEDFKGYE GCQALSEMIQ FYtERVMPQA (R1+R2 pan mutant) ENQDPDIKAH VNSLGENLKT RCHR FLPCENKSKA VEQVKNAFNK LQEKGIYKAM INYI LAYMTMKIRN GGSGGGGSGG SPGQGTQSEN SCTHFPGNLP NALRDLRDAF SRVKTFFQMK FQLDNLLLKL SRL*DEKGYR GCQATS‘MIQ t YELLVMPQA IKAH V'SLGENLKT LRLRLRRCTR FLPCEN<SKA VLQV<VAE'NK YKAM S LEDIEINYI E GGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN _ WKVDVALQSG NSQESVTEQD SKJSTYSLSS TLTLS<ADYE (HKVYACEVT HQGLSSPVTK SFNRGECGGG GSGGGGSGGGJ. GSGGGGSAST PLAP SS<STSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVVHKPSVTK VDKRVEPKSC PPCP APELLGGPSV FLFPP<PKDT LMISRTPEVT CVVVDVSH‘D PLVKENWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTIS<AK GQPREPQVYr1 *WTK NQVSLTCLVK GFYPSDIAVE WfiSNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALANHYTQKS LSLSPGK (SEQ ID NO: 30) M22A, SPGQGTQSEN SCTHFPGNLP NALRDLRDAF D41F:|inker:M22A SRVKTFEQMK FQRDNRRTKA. S.T*DE<GYT. GCQAT.S4MIQ *VMPQA (R1+R2 triple mutant) EVQDPDIKAH VNSLGENLKT LRLRLRRCHR FLPC3N<SKA VEQV<NAFNK INCORPORATEDITKREFERENCE(RULE206) 2017/038747 LQLKGIYKAM SLEDIEINYI LAYMTMKIRN GGSGGGGSGG SPGQGTQSEN GNLP NALRDLRDAF SRVKTFFQMK DQLDNLLLKE SLLEDFKGYL GCQATS MIQ tYK4‘VMPQA LNQDPDIKAH V'SLGLNLKT LRLRLRRCLR FLPC.LN<SKA VLQV<VAENK LQLKGIYKAM It'INYI .LAYMTMKIRN GGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN AKVQ LQSG NSQLSV"4QD SKJSTYSLSS TLTLS<ADYL ({KVYACLVT HQGLSSPV"K SFNRGLCGGGL GSGGGGSGGG GSGGGGSAST KGPSVFPLAP SS<STSGGHA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQmYIC NVNHKPSN"K PKSC D<THTCPPCP APLLLGGPSV FLFPP<P<D" LMISRTPLVT CVVVDVSH‘D PLVKENWYV) GVEVHNAK"K PREEQY STY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PILKTISKAK GQPRLPQVY" RPPSRLLW"K NQVSLTCLVK GFYPSDIAV.LLL' WESNGQ?ENN VLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALLNHYmQKS LSLSPG< (SEQ ID NO: 31) M22A, D41F:|inker:D41F SPGQGTQSEN SCTLFPGNLP NALRDLRDAF (R1+R2 triple mutant) SRVKTFTQMKL FQTDNTTTKL S‘TLDE<GYT GCQARSLMIQ w 4. L *VMPQA LNQDPD_KAH VNSLG.L LKT RCHR FLPCLN<SKA < {NAFNK YKAM SEFDIFINYI EAYWTW<IRN GGSGGGGSGG m FUNK (DIO<QGTQSEN SCTHFPGNLP NMLRDLRDAF SRV<TFFQMK FQED L m 4! 4. LDEKGYT GCQALSLMIQ ryma VMPQA LNQDPDIKAH VNSLGLNLKT HNF<k<w NLRRCHR FLPCEN<SKA VEQV<NAFNK LQE<GIYKAM SEFDIFINYI mV STWKIRN GGGGSGGGGS SVFI FPPSDLQLKS GTASVVCLLN 2W W RLA<VQ WKVDNALQSG NSQLSVHLQD S<DSTYSLSS TLTLS{A)Y.L W {KVYACLVT HQGLSS?V"K SFNRGLCGGG GSGGGGSGGG SAST KGPSVFPLAP SS<STSGG"A ALGCLVKDYF PLPVTVSWNS GALTSGVLTF PAVLQSSGLY SLSSVVTVPS SSLGTQmYIC SN"K VDKRVEPKSC DKTHTCPPCP APLLLGGPSV FLFPP<P<Dr1 LWISRTPLVT S{*D P‘VKtNWYVD GVLVHNA<fiK PRL'QYNSTY RVVSVLTVLH QDWLNGKLYK C(VSNKALPA PIEKTISXAK GQPREPQVYT ENTK NQVSLTCLVK GFYPSDIAVE WLSNGQ?*NN YK"TPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHLL ALLNHY"QKS LSLSPGK (SL 32) SPGQGTQSLN SC"{FPGNLP NALRDLRJAF M22AflmkenM22A FQWK DQmavmmmKR SWLEDFKGYE GCQALSEMIQ FYLERVMPQA D41F (R1+R2 triple ENQDPD_KAH NLKT LRLRLRRCHR FLPCENKSKA VEQVKNAFNK mutant) LQLKGIYKAM SLEDIEINYI LAYMTMKIRN GGSGGGGSGG SPGQGTQSEN SCTHFPGNLP NALRDLRDAF SRVKTFFQMK FQLDNLLLKE SLLEDFKGYL GCQATS‘MIQ t YEL‘VMPQA LNQDPDIKAH V'SLGLNLKT LRLRLRRCLR FLPCLN<SKA VLQV<VAENK LQLKGIYKAM SLEDIEINYI .LAYMTMKIRN GGGGSGGGGS RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESV“EQD SKJSTYSLSS TLTLS<ADYE (HKVYACEVT HQGLSS?V"K SFNRGLCGGGL GSGGGGSGGG GSGGGGSAST KGPSVFPLAP SS<STSGGHA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQmYIC NVNHKPSN"K VDKRVLPKSC D<THTCPPCP APLLLGGPSV FLFPP<PKD" PLVT CVVVDVSH‘D PLVKENWYVD GVEVHNAK"K PREEQY STY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PILKTISKAK GQPRLPQVY" EPPSR**W"K NQVSLTCLVK GFYPSDIAVL WESNGQ?ENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALLNHYmQKS < (SEQ ID NO: 33) SPGQGTQSEN GNLP NMLRDLRJAF D41F:|inker:M22A, D41F SRVKTFTQMKL FQLDNRRLKL STTLDE<GYT. LMIQ w 4. L *VMPQA (R1+R2 triple mutant) LNQDPD_KAH VNSLGL LKT LRLRLRRCHR FLPCLN<SKA < {NAFNK LQEKGIYKAM SEFDIFINYI EAYWTWKIRN GGSGGGGSGG m FUNK C110<QGTQSEN SCTHFPGNLP NALRDLRDAF SRV<TFFQMK FQRD RT.TKL m 4! 4. LDEKGYT GCQALSLMIQ EY144VMPQA LNQDPDIKAH VNSLGLNLKT HNF<k<w NLRRCHR FLPCEN<SKA VEQVKNAFNK LQEKGIYKAM SEFDIFINYI mV STWKIRN GGGGSGGGGS RTVAAPSVFI FPPSDLQLKS GTASVVCLLN 2W W RLA<VQ LQSG LSVHLQD S {DSTYSLSS TLTLS<ADYL W {KVYACLVT HQGLSSPVTK SFNRGECGGG GSGGGGSGGG GSGGGGSAST KGPSVFPLAP SS<STSGG"A KDYF SWNS VHTF PAVLQSSGLY SLSSVVTVPS SSLGTQmYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP GPSV FLFPPKPLDT LWISRTPLVT CVVVDVSH*D P‘VKtNWYVD INCORPORATEDITYREFERENCE(RULE206) GVEVHNA<TK PR44QYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE W‘SNGQ?*NN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMH? ALTNHYTQKS LSLSPGK (SEQ ID NO: 34) Expression of sc-IL-10 variant fusion proteins The genes were tically synthesized and supplied in pcDNA3.l expression vector (GeneArt), and transiently expressed in HEK293 cells using the Expi293 expression system (Life Technologies). Proteins were d using Protein A (GE Healthcare) with low pH elution and dialyzed against 2L 1X PBS 2 times.

The molecules were ed by SDS PAGE gel under reducing and ducing conditions. For reducing and non-reducing conditions, 2.5ug of protein was loaded onto an Any kD gel (Invitrogen) with a Precision Plus Protein oscope standard (Invitrogen) (MW range lOkD — 250 kD). The molecule was characterized by analytical gel filtration on an XBridge Protein BEH SEC column, 200A, 3.5 pm, 7.8 mm X 150 mm (Waters). The column was equilibrated and run at 0.9 ml/min with 100mM sodium phosphate pH 7.0 as a running buffer for all analyses. Purified samples (0.5mg/ml) were injected (15111) and eluted with a run time of 15 min.

Mouse PBMC Cytokine Release Assay In vitro bioactivity was assessed by evaluating the ability of our scIL-10 ucts to inhibit the production of TNFoc in LPS stimulated C57BL/6 mouse PBMCs (Bioreclamation).

For the assay, PBMCs cells were plated at 50,000 well in RPMI media containing 10% heat inactivated fetal bovine serum. Cells were incubated for 18 hours at 37°C, 5% C02 with 100 ng/mL LPS and varying concentrations of the scIL-lO ucts (R&D Systems). After 18 hours, TNFOL production was measured using V-Plex mouse TNFa MSD (Mesoscale Discovery). See Tables 5 and 6 below for IC50 values.

MC/9 Assay In vitro bioactivity was assessed by evaluating the ability of our scIL-lO constructs to stimulate proliferation of the mouse mast cell line MC/9 (ATCC CRL-8306). For the assay, MC/9 cells were plated at 10,000 cells/well in DMEM media ning 10% heat vated fetal bovine serum, 2 mM glutamine and 0.05 mM 2-mercaptoethanol. Cells were incubated for 72 hours at 37°C, 5% C02 with varying concentrations of human IL-10 (R&D Systems), RDB3515, RDB3516 or 9. After 72 hours, the cells were stained vuth CellTiter-Blue INCORPORATED BY REFERENCE (RULE 20.6) (Promega) for 4 hours at 37°C, 5% C02 according to the manufacturer’s protocol. Fluorescent measurements were taken at 560/590 nm. See Tables 5 and 6 below for EC50 values.

Table 5 thL-10 sclL-10:CL:CH1:Fc scIL-10:Fc (sclL-10:Fc), hinge truncation mutant 1 (scIL-10:Fc), hinge truncation mutant 2 (sclL-10:Fc), hinge truncation mutant 3 |87A:|inker:l87A(vlL10 mutant) |87A:|inker:wt|L-10 (vlL10 ) 0:|inker:|87A (vlL10 mutant) D41F:|inker:wt|L-10 (R1 mutant) 2200 wtlL-10:IinkerD41F (R1 mutant) 7600 D41F:|inker:D41F (R1 mutant) No activity M22A:Iinker:wt|L-10 (R2 mutant) 610.4 :Iinker:M22A (R2 mutant) 844.4 M22A:Iinker:M22A (R2 mutant) 741.1 M22A:Iinker:D41F (R1+R2 mutant) . 470 thL-10:Iinker:M22A, D41F (R1+R2 mutant) . 2534.6 D41F:|inker:M22A (R1+R2 mutant) . >> 10000 inker:D41F (R1+R2 ) . >> 10000 M22A, D41F:|inker:M22A, D41F (R1+R2 pan mutant) No activity M22A, D41F:|inker:M22A (R1+R2 triple mutant) >> 10000 M22A, D41F:|inker:D41F (R1+R2 triple mutant) No activity M22A:Iinker:M22A, D41F (R1+R2 triple mutant) >> 10000 D41F:|inker:M22A, D41F (R1+R2 triple mutant) No activity sclL-10:CL:CH1:Fc(sc|L10 5aa linker) . 85.7 sclL-10:CL:CH1:Fc(sc|L10 3aa ) 50 INCORPORATED BY REFERENCE (RULE 20.6) O:CL:CH1:FC wtlL-10:Iinker:D41A (R1 mutant) 2383-333 . 1430 thL-10:Iinker:M22A, D41A (R1+R2 D41A:Iinker:M22A (R1+R2 mutant) ND M22A:Iinker:D41A (R1+R2 mutant) ND wtlL-10:Iinker:D41N (R1 mutant) 14 thL-10:|inker:M22/-\, D41N (R1+R2 988.75 mutant) D41N:Iinker:M22A (R1+R2 mutant) 2780 604-3478 M22A:Iinker:D41N (R1+R2 mutant) As shown in Table 5, the ratio for WT IL-10 was ~11. The ratio for SEQ ID NO: 12 was 1000, showing that just by building the 0 sequence on the CL:CH1:Fc scaffold, the anti-inflammatory window is sed. The following ments were conducted with various configurations of scIL-10 molecules of Formula 1 ing unsubstituted scIL-10, scIL-10 variants and LINKER lengths of various sizes on the CLzCleFc scaffold.

Experiments were conducted using the constructs of Tables 5 and 6 to explore the s unsubstituted scIL—10 and scIL-10 variants that disrupt the scIL-lO interfaces with ent combinations of the two IL-10R1 and two 2 receptor chains from the scIL-10 heteropentameric signaling complex. Mutations that disrupt either one of the two IL-10R1 interfaces SEQ ID NOS: 20, 21, 37 and 41 as illustrated in slightly weaken the anti- inflammatory potency, while significantly weakening the immunostimulatory potency, resulting in an increase in the anti-inflammatory window size. ucing a double mutation that simultaneously disrupts both IL-10R1 interfaces (SEQ ID NO: 22) results in a construct with no measurable anti-inflammatory or immunostimulatory activities. This demonstrates that in order for scIL-10 to signal via the IL-10 receptor, it must be able to recruit at least 1 IL-10R1 receptor chain. Since the IL- 10R1 receptor chain is known to be the “high affinity” receptor chain (binding more tightly to IL-10 than IL-10R2 does), it is likely that mutations that simultaneously disrupt both 1 binding interfaces would eliminate or significatly weaken the ability of scIL-10 to bind to the IL-10 receptor, resulting in no signal transduction at all.

Mutations that disrupt either one of the two IL-10R2 interfaces (SEQ ID NOS: 23 and 24), as rated in demonstrate no change in the anti-inflammatory potency, INCORPORATED BY REFERENCE (RULE 20.6) while showing a slight increase in the immunostimulatory potency, resulting in slightly decreased anti-inflammatory window sizes. Introducing a double-mutant that simultaneously disrupts both IL-10R2 interfaces (SEQ ID NOS, 29) leads to a loss in potency for both antiinflammatory and immunostimulatory activities, resulting in a construct with an anti- inflammatory window size r to that of the IL-10R2 ace single mutants, which is slightly reduced relative to the native scIL-lO construct. This result demonstrates that mutations that disrupt the IL-10R2 interface do not alone have the potential to expand the anti-inflammatory window of scIL-IO.

Mutations that aneously disrupt one of the IL-lORl and one of the IL-10R2 interfaces were explored as illustrated in Mutations located in the IL-lORl and IL- IORZ sites from the same side of the scIL-lO fused dimer (SEQ ID NOS: 26 AND 27) demonstrate weakened potency for both anti-inflammatory and immunostimulatory activities; one of those combinations (SEQ ID NO: 27) displays a significantly increased anti- inflammatory window size. Mutations located in an IL-lORl interface and an IL-IOR2 ace from opposite sides of the scIL-IO fused dimer (SEQ ID NOS: 28 and 29) display weakened anti-inflammatory potency, and no able immunostimulatory activities at the concentrations tested. Therefore, they display extremely large anti-inflammatory windows.

Since IL-10 receptor signal transduction requires IL-IORI and IL-10R2 to be clustered following IL-IO binding, these data te that the l strategy for attenuating timulatory activity (and thereby sing the nflammatory window) is to target both of the pairs of IL-lORl/IL-10R2 receptor chains. Since the IL-IORI interface scan revealed that signaling es that at least one I interface be competent for binding, it is necessary to target the IL-10R2 interface on the opposite side of the scIL-IO fused dimer, to effectively t both pairs of IL-lORl/IL-IOR2 receptor chains that cluster upon scIL- lO binding. This pattern of mutations more dramatically modulates scIL-lO bioactivity on cells that mediate immunostimulation, while the cells that mediate the anti-inflammatory effects remain quite ive to scIL-IO signaling.

INCORPORATED BY REFERENCE (RULE 20.6) Example 3 Varying linker length of scIL- 10 The scIL-lO of Formula 1 wherein LINKER length is varied are fused to a single chain Fc linker of Formula 2 wherein L1 is CL-CHl-Fc as per Formula 3. The amino acid sequences of each full length scIL-lO-Ll-HINGE-Fc fusion variant protein synthesized is found in Table 7.

For expression in mammalian cells, the N-terminal leader sequence of SEQ ID NO: 48 was added to each of the protein sequences found in Table 7.

The amino acid sequences of each fusion protein are found in Table 7. Expression of peptides are as described in Example 2. Bioactivity of was tested in a mouse PBMC cytokine release assay and an MC/9 assay as described in Example 2. The results are found in Table 5 of Example 2. The results show that decreasing the size of the linker reduces the size of the anti-inflammatory , implying that the linker length affects the strength of the lL-lORl and IL-10R2 interfaces in ways that reduce the selectivity for anti-inflammatory potency over immunostimulatory potency.

Table 7 Description Amino Acid Sequence Unsubstituted 0(5aa SPGQGTQSEN SCTHFPGWLP VMLRDLRDAF |inker):CL:CH1:Fc) SRVKTFFQWK DQFDNFLFKfi SRFfiDE<GYR GCQAESfiMIQ EYE A ENQDPDIKAH VNSLGENLKT ARLRLRRCHR KSXA VEQVKNAFNK LQEKGIYKAM SLFDIFIWYI LAYMTM<IRN GGSGGSPGQG TQSEVSCTHF ?GVLPNWLRD LRDAFSRVKT FFQMKDQFDN TLKK‘SKL*D FKGYLGCQAL YLEE VWPQAENQDP DIKAHVNSLG ENLKTLQLQL LPCE NKSKAVEQVK AFNKLQEKG jEDI EINYIjAYWT GGGS GGGGSRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN ALQSGNSQES V"EQDSKDST YSLSSTLTLS <ADYE<HKVY ACEV"HQGLS SPVTKSFNRG ECGGGGSGGG GSGGGGSGGG GSAST<GPSV FPLAPSS<ST SGGTAALGCL VKDYFPEPVT VSWNSGALTS AVLQ SSGLYSLSSV SLGT Q"YICNVVHK PSVHKVDKRV EPKSCD<THT CPPCPAPELL GGPSVFLFPP {PKDTLMISR TPEVTCVVVD VSHiDPiVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN IS<AKGQPR3 PQVYTFPPSR * *WTKNQVSL FYPS QPENNYK”TP PVLDSDGSFF VFSC GK (SEQ :D SASP O: 39) SCTAFPGNAP Domoummm<r IT'MZZIT'YSKLTVDKS scHxIOOfFonnMal SPGQGTQSEN MLRDLRDAF wherein LINKER is SRVKTFFQM< LFFDFKGYR 3 GCQALSFMIQ FYLFFVMPQA I<Al VNSLGENL<T RLRLRRCTR FLPCENKS<A EQVKNAFNK amino acid linker LQEKGIYKAM SEFDIFINYI EAYMTMKI?N GGGSPGQGTQ ENSCTHFPG NLPNMLRDLR DAFSRVKTFF QMKDQLDNAL EDFK ulorym PQAEVQDPDI KAHVNSLGEN {RFLPCENK avu LKTLRLRLRR FNKLQEKGIY KAMSEFDIFI Homm<YLGCQAASE SKAVEQVKNA uYIEAYMTWK RNGGGGSGG GGSRTVAAPS VFIFPPSDEQ L<SGTASVVC LLVNFYPREA {VQWKVDVAL QSGNSQfiSVr1 SmYS LSSTLTLSiA )YEKTKVYAC EVTHQGLSSP VTKSFNRGEC GGGGSGGGGS GGGGSGGGGS ASTKGPSVFP STSG CLVK DYFPEPVTVS WVSGALTSGV {"FPAVLQSS GLYSLSSVVT VPSSSLGTQr1 YICNVNHKPS V"KVDKRVEPJ. <SCD<THTCP PCPAPELLGG {DTLWISRTP EVTCVVVDVS KtNW YVDGVEVHNA STYRVVSVLT VLHQDWLNGK SNKA LPAPZEKTIS VYmbPPSRii W"KNQVSLTC LVKGFYPSDI AV4WJSNGQP INCORPORATEDITKREFERENCE(RULE206) ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MH3A3HNHYT QKSLSLSPGK (SEQ ID NO: 36) Example 4: Modulating the anti-inflammatory window of scIL-10 via steric crowding.

The amino acid sequences of each sclL-lO fusion protein used in this experiment are mmflnfhwRS.BmmmbnofimfiflaiumdmammhnEmmmRZ.BmmuWWof peptides was tested in a mouse PBMC cytokine release assay and an MC/9 assay as bed in Example 2. The results are found in Table 5 of Example 2. The results show that as the hinge is shortened, the nflammatory window ses in size. Without being limited to any particular theory, this implies that hinge tion increases steric crowding n two scIL-10 moieties, resulting in modulation of the IL-10R1 and IL—10R2 aces, which translates to altered anti-inflammatory and immunostimulatory potencies.

Table 8 Description: Amino Acid Sequence 0:Fc MYRMQLLSC: VTNS SPGQGTQSEN SCTHFPGNLP NMLRDLRDAF FQM< DQTJNTMH SELLDE<GYR GcoAis *MIQ EYTH 4VMPQA ENQDPDIKAH \NSLGENLKr1 LRL LRRCHR FLPCENKSKA VEQVKNAFNK LQLKGIYKAM SLEDIEIVYI <IRN GGSGGSPGQG TQSLVSCTHF PGVLPNWLRD LRDAFSRVKr1 FFQW<DQLDN TmTTK'ST.RLD EKGYLGCQAL SEMIQFYLEE VMPQAENQDP DIKAJVNSLG RLRL RRCH.2FLPCE NKSKAVEQV< NAFVKLQEKG IYKAMSLEDI EINYILAYW"1. MKIRVEPKSS DKTHTCPPC? APELLGGPSV PKDr1 LMISRTPEVr1 CVVVDVSHI‘D PEVKFNWYVD GVEVHNAKT< PREEQYVSHY RVVSVLTVLH QDWLHGKEYK CKVSNKALPA P:E<TIS{A< GQPRLPQVYr1 TPPSRL'LWTK NQVSLTCLVK GFYPSDIAVA. WLSVGQPLN YKTTPPVLDS DGSFFLYSKL TVD<SRWQQG HVFSCSVMHE ALHVHYTQKS LSLSPG< (SEQ ID V0: 13) (sclL-10:Fc), 4AA SPGQGTQSEVJ. SCTTFPGVLP NMLRDLRDAF hinge truncation SRVKTFFQW< DQT.DNT.T.T.K* SRRLDE<GYE GCQALSLMIQ EYEL VMPQA EVQDPDIKAT VNSLGENLKr1 LRL LRRCHR FLPCEV<S<A <EQV<NAFN< LQEKGIYKAM SEFDIFINY: EAYM3M_<IRN GGSGGSPGQG HQSENSCTHF mGNLPNMLRD LRDAFSRVKr1 FFQH<DQLDN T...TT (*STJHD W (GYLGCQAL w *MIQtYlii NQDP DIKATVVSLG LNL<"LRLRL wRCHRFLPCL ZKSKAVEQVK NAFVKLQEKG IYKAMSEFDI FINYTEAYWT ZKIRVSDKTH aCPPCPAPEL LGGPSVFLFP PKP<DTLMIS RTPEVTCVVV DVSHLDPLV< WNWYVJGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQD'L NGKEYKCKVS Z IE< TIS<A<GQPR EPQVYTLPPS 2EEW”KNQVS GFYP m DIAV‘W‘SV Y<TT PPVLDSDGSF FLYS<LTV3K SRWQQGNVFS HEALTV HYTQKSLSLS PGK (SEQ ID NO: 14) (sclL—10:Fc), 7 aa m PGQGTQSEV SCTTFPGVLP VMLRDLRDAF hinge truncation m RVKTFFQMK DQLDNLLLKE SLLEJFKGYL GCQALSEMIQ MPQA mVQDPDIKAT VWSLGE LKT LRLRLRRCHR KSKA LQVKNAFN< H SLEDIEIVYI .LAYM"M<IRN GGSGGSPGQG QSLNSCTHF m LRDAFSRVKT FFQM<DQLDN LLLKESLLED {GYLGCQAL m L VWPQA? QDP DIKATVVSLG LNLKTLRLRL LEDI EINYILAYMT S/unji—ZI<I RCTRFLPC_3 z NAFVKLQEKG {IRNTHTCP PSVFLFPPKP (DTLWISRTP EVTCVVVDVS HEDPEVKFNW KTKPR**QYN STYRVVSVLT VLHQDWLNGK LYKCKVSNKA INCORPORATED BY REFERENCE (RULE 20.6) LPAPILKTIS KAKGQPRLPQ VYTLPPSRA. 4 MTKNQVSLTC LVKGFYPS DI AVEWESNGQ? ENNYKTTPPV FFLY SKLTVDKSRW QQGWFscsv MHLALINHYT QKSLSLSPG< (S 3Q ID NO: 15) :Fc), 10aa SPGQGTQSEN SCTHFPGVLP RDAF hingetruncafion. SRVKTFFQMK DQRDNRKEKL SRRLDEKGYP GCQATSLMIQ PYEL'VMPQA ENQDPDIKAH VNSLGENLKT LRLRLRRCflR FLPCENKS_{A NAFNK YKAM SEEDIFIVYI EAYWTMKIRV GGSGGSPGQG TQSEVSCTHF PGVLPVMLRD LRDAFSRVK" FFQWKDQRDV K*SI.T.*D t<GYLGCQAL SEWIQFYLEE VMPQAENQDP DIKAHVNSLG ENLKTLRLRL RRCHRFLPCE NKSKAVEQVK NAFNKLQEKG IYKAMSEFDI FINYIEAYMT THTCP PCPAP.3LLGG PSVFLFPPKP KDTLMISRTP LVTCVVVDVS HLDPLVKENW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPILKTIS {AKGQPREPQ VY"I.PPSR** SLTC LVKGFYPSDI AVLWLSNGQ? TPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALANHYT QKSLSLSPGK (SEQ ID NO: 16) Example 5- scIL-10 Experiments were conducted with scIL-10 of Formula 1 wherein LINKER was of varying lengths. The amino acid sequences synthesized for these experiments are shown in Table 9. Expression of SEQ ID NOS: 45 and 46 is as described in Example 2. Bioactivity of SEQ ID NOS 45 was tested in an MC/9 assay as described in Example 2. The data showed that the value for SEQ ID NO: 45 in the MC/9 was 5.6 pM.

Bioactivity of SEQ ID NOS 45 and 46 will be further tested in a mouse PBMC cytokine release assay and an MC/9 assay as described in Example 2. The results will show that the scIL-IO moiety, absent any fusion r, demonstrates highly potent bioactivity, consistent with the trends observed for O Fc fusion proteins.

Table 9 Description Amino Acid Sequence scIL-IO with 5 SPGQGTQSEV SCTHFPGNLP NMLRDLRDAF amino acid linker SRV<TFFQW< DQT.)NT.T.T.K* SDD*DP<GYE GCQALS‘MIQ EYIH‘ EvQJPDIKAi VNSLGLNLKr1 LRL LRRCHR FLPCEN<S<A AFN< QEKGIYKAM SEFDIFINY: EAYMTM_<IRN GGSGGSPGQG PGNLPNMLRD LRDAFSRVKr1 FFQW<DQLDN TMTTKLSTILD S *MIQPYLJ. 4 VMPQATNQDP DIKAIVVSLG LNLKTLRLRL EQVK IYKAMSEFDI FINYIEAYWT ID NO: 45) scIL-IO with 10 SPGQGTQSEN SCTflFPGNLP NMLRDLRDAF amino acid linker SRV<TFFQM< DQT.DNT.T.T.K4 SIJ. A. DE<GYT LMIQ EVQDPDIKAH VWSLGENLKT LRL LRRCHR FLPCENKSKA LQL<GIYKAM SLtDItINYI LAYWTM<IRN GSGG SCTIFPGNL? NWLRDLRDAF FQMK DQIWDNITIK*.

GCQALSEMIQ FYLEEVMPQA ENQDPDIKAH VNSLGENLKT FLPCENKSKA VEQVKNAFNK LQE<GIYKAM SEFDIFINYI (S 3Q ID NO: 46) INCORPORATED BY REFERENCE (RULE 20.6) The scIL-lO of Formula lwas fused to a mucin domain linker comprising a tandem repeat of MUC 20 which in turn was fused to an Fc domain. The amino acid sequence of the lO (5aa linker))-(mucin )-Fc is found in Table 10. For expression in mammalian cells, the N-terminal leader sequence of SEQ ID NO: 48 was added to the protein found in Table 11.

The amino acid sequences of each fusion protein are found in Table 10. Expression of peptides are as described in e 2. Bioactivity of was tested in a mouse PBMC cytokine release assay and an MC/9 assay as described in Example 2. The results are found in Table 11. The results show that the bioactivities of scIL-lO Fc fusion proteins are consistent regardless of the composition of the linker domain connecting the scIL-lO and Po domains.

TABLE 10 Description Amino Acid Sequence (scIL-10(5aa WYRMQLLSCT ALSLALVTNS SPGQGTQSEV SCTHFPGNLP NWLRDLRDAF linker))-(mucin m QMK DQLDNALLKE SLLEDFKGYA GCQAASEMIQ FYLEEVMPQA MVQDPDIKAT VNSLGENLKr1 LRLRLRRCHR FLPCENKSiA V.3QVKNAFN< linker)—Fc YKAM SEFDIFINYI EAYMTMKIRV GGSGGSPGQG TQSENSCTHF mGNLPNMLRD RVKr1 FFQMKDQEDV th srmD t<GYLGCQAL w MMIQEYT dd VMPQAENQDP DIKAHVNSLG ENLKmLRLRL R?ClRFLPC.E Z.(SKAVEQVK NAFNKAQEKG IYKAMSEFDI FINY_EAYMT MKI.RNSGSGG ASSESSASSD GPHPVITESR ASSESSASSD TESR EPKSSD<THT CPPCPAPELL LFPP KPKD"LMISR TPEV"CVVVD VSH4DP VKt NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV G<EYKCKVSN KALPAPIEKT IS<AKGQPRE PS? *.*. TCLVKGFYPS DIAV4W45NG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK (SEQ ID V0: 52) Table 11 SEQ ID NO PMBC MC/9 '11PM 21PM 1909 INCORPORATED BY REFERENCE (RULE 20.6) The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby orated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to red features thereof, it will be tood by those skilled in the art that various changes in form and s may be made therein without departing from the scope of the invention encompassed by the appended claims. It should also be understood that the various es of the invention described herein are not mutually exclusive and that features may be combined in whole or in part in accordance with the invention.

INCORPORATED BY REFERENCE (RULE 20.6)

Claims (43)

1. Use of a scIL-10 in the manufacture of a medicament for ent to modulate the immunostimulatory and anti-inflammatory properties of IL-10 in a patient in need of IL-10 therapy, wherein the treatment ses the administration of scIL-10 comprising an amino acid sequence arrangement from N-terminus to C-terminus in accordance with a 1: (first monomer subunit)-LINKER-(second r subunit) Formula 1 wherein the first monomer subunit and second monomer subunit may be independently selected from: SEQ ID NO: 1; or SEQ ID NO: 1 comprising at least one amino acid substitution selected from: amino acid position 22, amino acid position 41, amino acid position 87, and any combination thereof, with the proviso that at least one of the first monomer subunit or the second monomer t comprises the at least one amino acid substitution; wherein scIL-10 modulates the immunostimulatory or anti-inflammatory properties as ed to wtIL-10, wherein LINKER is an amino acid linker of between about 1 and about 100 amino acids in length; and wherein scIL-10 is optionally covalently attached to a fusion partner.

2. The use of claim 1 wherein LINKER is 5-15 amino acids in length.

3. The use of claim 1 wherein the amino acid tutions comprise the substitution of amino acids of scIL-10 that interface with IL-10R1, IL-10R2 or amino acids that interface with both IL-10R1 and IL-10R2.

4. The use of claim 1 wherein the amino acid substitutions comprise substitutions of amino acids selected from: methionine at position 22 and aspartic acid at position 41 or any combination thereof.

5. The use of claim 4 wherein aspartic acid at position 41 is substituted on the first monomer subunit or on the second monomer subunit but not both monomer subunits.

6. The use of claim 5 wherein methionine at position 22 is substituted on only one r subunit that is not the same r subunit comprising the substitution of aspartic acid at position 41.

7. The use of claim 1 wherein the amino acid substitution at on 87 comprises isoleucine to alanine .

8. The use of claim 3 wherein the amino acid substitutions are selected from: methionine at position 22 to alanine (M22A); aspartic acid at position 41 to asparagine ; aspartic acid at position 41 to alanine (D41A); aspartic acid at position 41 to phenylalanine (D41F).

9. The use of claim 1 comprising a fusion partner wherein scIL-10 is fused to the hinge region IgG1.

10. The use of claim 1 comprising a fusion partner wherein scIL-10 is fused to a modified hinge region if IgG1 wherein the modification to the hinge region is the deletion of between 1 and 10 amino acids from the hinge region of IgG1.

11. The use of claim 1 comprising a fusion partner wherein scIL-10 is fused to the hinge region of IgG1 via a mucin linker.

12. The use of claim 11 wherein the mucin linker ses an amino acid sequence that is a tandem repeat of MUC20.

13. The use of claim 1 comprising a fusion r wherein scIL-10 is fused to a single chain Fc linker wherein the fusion protein has the sequence of Formula 2 (scIL-10)-L1-HINGE-Fc Formula 2 wherein, L1 is a linker having the following arrangement from amino-terminus to carboxyterminus L2-CL-L3-CH1-L4 or L2-CH1-L3-CL-L4 wherein, L2 and L4 are ndently polypeptide linkers or are independently absent; L3 is a polypeptide linker; CL is a constant region polypeptide of an immunoglobulin light chain; and CH1 is a constant region polypeptide from a CH1 domain of an immunoglobulin heavy chain; HINGE is a hinge sequence of an immunoglobulin or is absent with the proviso that if HINGE is absent, L4 is present; and Fc is the carboxy-terminus of an immunoglobulin or any active fragment or derivative thereof.

14. An scIL-10 polypeptide comprising an amino acid sequence arrangement from N- us to C-terminus in accordance with Formula 1: (first monomer subunit)-LINKER-(second monomer subunit) Formula 1 wherein the first monomer subunit or the second monomer subunit may be independently selected from: SEQ ID NO: 1; or SEQ ID NO: 1 comprising at least one amino acid substitution selected from: amino acid position 22, amino acid position 41, amino acid position 87, and any combination thereof, with the proviso that at least one of the first monomer subunit or the second r subunit comprises the at least one amino acid substitution; and n LINKER is an amino acid linker of between about 1 and about 100 amino acids in length.

15. The polypeptide of claim 14 wherein LINKER is 5-15 amino acids in length.

16. The polypeptide of claim 14 wherein the amino acid substitutions se the tution of amino acids of scIL-10 that ace with 1, IL-10R2 or amino acids that interface with both IL-10R1 and IL-10R2.

17. The polypeptide of claim 14 wherein the amino acid substitutions se substitutions of amino acids selected from: nine at position 22 and aspartic acid at on 41 or any ation thereof.

18. The polypeptide of claim 17 wherein aspartic acid at position 41 is substituted on the first monomer subunit or on the second monomer subunit but not both monomer subunits.

19. The polypeptide of claim 18 wherein nine at position 22 is substituted on only one monomer subunit that is not the same monomer subunit comprising the substitution of aspartic acid at position 41.

20. The polypeptide of claim 14 wherein the amino acid substitution at position 87 comprises isoleucine to alanine (I87A).

21. The polypeptide of claim 16 wherein the amino acid substitutions are selected from: methionine at position 22 to e (M22A); aspartic acid at position 41 to asparagine (D41N); aspartic acid at position 41 to alanine (D41A); aspartic acid at position 41 to alanine (D41F).

22. The polypeptide of claim 14 comprising a fusion partner wherein scIL-10 is fused to the hinge region IgG1.

23. The polypeptide of claim 14 comprising a fusion partner wherein scIL-10 is fused to a modified hinge region if IgG1 wherein the modification to the hinge region is the deletion of between 1 and 10 amino acids from the hinge region of IgG1.

24. The polypeptide of claim 14 comprising a fusion partner wherein scIL-10 is fused to the hinge region of IgG1 via a mucin linker.

25. The polypeptide of claim 24 wherein the mucin linker comprises an amino acid sequence that is a tandem repeat of MUC20.

26. An scIL-10 ptide of claim 14 sing a fusion partner wherein scIL-10 is fused to a single chain Fc linker wherein the fusion protein ses an amino acid sequence of a 2 (scIL-10)-L1-HINGE-Fc wherein, L1 is a linker having the following arrangement from amino-terminus to carboxyterminus L2-CL-L3-CH1-L4 or -L3-CL-L4 wherein, L2 and L4 are independently polypeptide linkers or are independently absent; L3 is a polypeptide linker; CL is a constant region polypeptide of an immunoglobulin light chain; and CH1 is a constant region polypeptide from a CH1 domain of an immunoglobulin heavy chain; HINGE is a hinge sequence of an immunoglobulin or is absent with the proviso that if HINGE is absent, L4 is present; and Fc is the carboxy-terminus of an immunoglobulin or any active fragment or derivative thereof.

27. The polypeptide of claim 26, wherein CL, CH1, HINGE and Fc are at least 90% identical to the CL, CH1, hinge and Fc regions respectively of human IgG1.

28. The polypeptide of claim 26, wherein L3 is a polypeptide linker having the amino acid sequence (GGGGS)n wherein n is 1-5.

29. The polypeptide of claim 26, n L2 is present and is a polypeptide linker having the amino acid sequence (GGGGS)n wherein n is 1-5.

30. The polypeptide of claim 26, wherein L4 is present and is a polypeptide linker having the amino acid sequence (GGGGS)n wherein n is 1-5.

31. The polypeptide of claim 26, wherein HINGE and L2 are present and L4 is absent.

32. The polypeptide of claim 26, wherein HINGE, L2 and L4 are present.

33. The polypeptide of claim 26, wherein HINGE is absent and L4 is present.

34. The polypeptide of claim 26, wherein HINGE is absent and L2 and L4 are t.

35. A dimerized complex comprising the polypeptide of claim 26 wherein L1 is a linker having the following ement from amino-terminus to carboxy-terminus: L2-CL-L3-CH1-L4.

36. A polypeptide of claim 26 selected from the group ting of: SEQ ID NOs: 20-21 and SEQ ID NOS: 37-44

37. A polypeptide of claim 26 selected from the group consisting of: SEQ ID NOS: 17, 18 and 19.

38. The polypeptide of claim 14, n the linker is not GSGG (SEQ ID NO: 3); and wherein scIL-10 is covalently attached to a fusion partner.

39. A polypeptide of claim 38 comprising a fusion partner wherein scIL-10 is fused to a single chain Fc linker wherein the fusion protein comprises an amino acid sequence of Formula 2 (scIL-10)-L1-HINGE-Fc Formula 2 wherein, L1 is a linker having the following arrangement from amino-terminus to carboxyterminus L2-CL-L3-CH1-L4 or L2-CH1-L3-CL-L4 wherein, L2 and L4 are independently polypeptide linkers or are independently absent; L3 is a polypeptide linker; CL is a nt region polypeptide of an immunoglobulin light chain; and CH1 is a constant region polypeptide from a CH1 domain of an immunoglobulin heavy chain; HINGE is a hinge sequence of an immunoglobulin or is absent with the proviso that if HINGE is absent, L4 is present; and Fc is the carboxy-terminus of an immunoglobulin or any active nt or derivative thereof.

40. The polypeptide of claim 14, wherein scIL-10 is covalently attached to a fusion partner that comprises a mucin domain polypeptide.

41. The use of claim 1 wherein the polypeptide comprises a sequence selected from the group consisting of: SEQ ID NOS: 17-21, 23-29, 31, 33, and 37-44.

42. The ptide of claim 38 wherein the fusion partner comprises an IgG1 Fc region including a hinge region.

43. The polypeptide of claim 42 wherein 1-10 amino acids have been d from the hinge region.