Interleukin- 10 fusion proteins and methods of use

Abstract

Disclosed herein are IL10 fusion proteins which are myeloid selective. Methods of production and methods of therapeutic use of IL 10 fusion proteins are also disclosed.

Description

Attorney Docket No. 2019802-0011INTERLEUKIN- 10 FUSION PROTEINS AND METHODS OF USECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Application Serial No. 63 / 736,207 filed on December 19, 2024, the disclosure of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Interleukin- 10 (IL10) is a pleiotropic immunomodulatory cytokine produced byM , B, NK, and T cells (CD4+, CD8+, and Tregs). IL 10 binds as a noncovalent homodimer with high affinity to the IL 10 receptor alpha (ILlORa) leading to recruitment of the IL 10 receptor beta (IL1OR0). Receptor binding activates a complex signaling cascade, including phosphorylation of STAT3 and STAT1. Signaling through this pathway can lead to both antiinflammatory and pro-inflammatory effects on various immune cell subsets where the receptor is expressed. Pro-inflammatory effects include expansion, activation, and cytolytic potentiation of primed CD8+T cells and NK cells. Comparatively, anti-inflammatory effects include suppression of myeloid cytokine production and priming capacity.

[0003] Therefore, there is a need to develop new IL10 therapeutics for treating inflammatory diseases, which selectively suppresses myeloid cytokine production and induces tolerogenic myeloid cells, thereby providing a sustained response for patients in need thereof.SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on December 08, 2025, is named 2019802-0011, and 16,536 bytes in size.113165576vlAttorney Docket No. 2019802-0011SUMMARY

[0005] The present disclosure provides strategies, compositions, methods, and kits, related to IL10 fusion proteins. In some embodiments, IL10 fusion proteins described herein have an IL10 polypeptide with a spacer insertion sequence. Among other things, it is an insight of the present disclosure that the disclosed strategies, compositions, methods, and kits provide beneficial effects relative to IL10 fusions proteins that have a wild-type IL10 polypeptide (i.e., an 1L10 polypeptide without a spacer insertion sequence).

[0006] Interleukin- 10 (IL10) is an immunomodulatory cytokine produced by immune cells, that triggers complex signaling pathways that lead to anti-inflammatory and pro- inflammatory responses. The present disclosure provides various methods and compositions of IL10 fusion protein, which can be employed to modulate the immune system.

[0007] In some embodiments, the present disclosure provides an IL10 fusion protein comprising an amino acid sequence at least about 99% identical to SEQ ID NO: 1.

[0008] In some embodiments, the present disclosure provides an IL10 fusion protein consisting of a first polypeptide consisting of an amino acid sequence at least about 99% identical to SEQ ID NO: 1 and a second polypeptide consisting of an amino acid sequence at least about 99% identical to SEQ ID NO: 1, wherein the first polypeptide and the second polypeptide each comprise an Fc region, and wherein the first polypeptide and the second polypeptide are associated with each other by at least one covalent disulfide bond and / or at least one non-covalent interaction between the Fc region of the first polypeptide and the Fc region of the second polypeptide.

[0009] In some embodiments, the present disclosure provides an IL10 fusion protein comprising the amino acid sequence as set forth in SEQ ID NO: 1

[0010] In some embodiments, the present disclosure provides an IL10 fusion protein consisting of a first polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 1 and a second polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 1, wherein the first polypeptide and the second polypeptide each comprise an Fc region, and wherein the first polypeptide and the second polypeptide are associated with each other by at213165576vlAttorney Docket No. 2019802-0011 least one covalent disulfide bond and / or at least one non-covalent interaction between the Fc region of the first polypeptide and the Fc region of the second polypeptide.

[0011] In some embodiments, the present disclosure provides an IL10 fusion protein comprising: (a) an IL 10 polypeptide comprising an amino acid sequence at least about 95% identical to SEQ ID NO: 2; (b) an Fc polypeptide comprising an amino acid sequence at least about 95% identical to SEQ ID NO: 3; and (c) a linker operably linked to the IL 10 polypeptide and to the Fc polypeptide, wherein the C-terminus of the Fc polypeptide is linked to the N- terminus of the linker and wherein the N-terminus of the IL 10 polypeptide is linked to the C- terminus of the linker.

[0012] In some embodiments, the IL 10 polypeptide comprises an amino acid sequence at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 2. In some embodiments the IL10 fusion protein comprises the amnio acid sequence according to SEQ ID NO: 2. In some embodiments the IL 10 fusion protein consists of the amino acid sequence according to SEQ ID NO: 2.

[0013] In some embodiments, the Fc polypeptide comprises an amino acid sequence at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 3. In some embodiments, the Fc polypeptide comprises the amnio acid sequence according to SEQ ID NO: 3. In some embodiments, the Fc polypeptide consists of the amino acid sequence according to SEQ ID NO: 3.

[0014] In some embodiments, the linker consists of 14 amino acids, 15 amino acids, or 16 amino acids. For example, the linker comprises the amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the IL10 fusion protein further comprises a heterologous moiety.

[0015] In some embodiments, the 1L10 fusion protein of the present invention is more myeloid selective than an IL10 polypeptide consisting of SEQ ID NO: 6 or an IL10 polypeptide consisting of SEQ ID NO: 8.

[0016] In some embodiments, the IL 10 fusion protein of the present invention is a monomer. In some embodiments, the IL10 fusion protein is a dimer comprising two monomers, and wherein the two monomers are associated with each other via their Fc regions. In some313165576vlAttorney Docket No. 2019802-0011 embodiments, the Fc regions of the two monomers are associated with each other by covalent disulfide bonds and / or non-covalent interactions. In some embodiments, the IL10 fusion protein is a dimer, wherein it comprises two identical monomers, and optionally, each of the monomers comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the IL10 fusion protein is a dimer, wherein it comprises two different monomers, and optionally, each of the monomers comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] In some embodiments, the present disclosure provides a composition comprising the IL10 fusion protein disclosed herein.

[0018] In some embodiments, the present disclosure provides a nucleic acid that encodes the IL10 fusion protein disclosed herein.

[0019] In some embodiments, the present disclosure provides a vector comprising the nucleic acid disclosed herein.

[0020] In some embodiments, the present disclosure provides a host cell comprising the nucleic acid disclosed herein. Optionally, the host cell is a mammalian cell.

[0021] In some embodiments, the present disclosure provides a pharmaceutical composition comprising (a) the IL10 fusion protein disclosed herein, the composition disclosure herein, the nucleic acid disclosed herein, the vector disclosed herein, or the host cell disclosed herein; and (b) a pharmaceutically acceptable excipient.

[0022] In some embodiments, the present disclosure provides a kit comprising the fusion protein disclosed herein and instructions for administering the fusion protein to a subject in need thereof.

[0023] In some embodiments, the present disclosure provides a method of producing the 1L10 fusion protein disclosed herein, comprising culturing the host cell disclosed herein under suitable conditions and recovering the fusion protein. In some embodiments, the host cell is a mammalian cell.

[0024] In some embodiments, the present disclosure provides a method of treating a disease or disorder a subject in need thereof, comprising administering to the subject an effective amount of the fusion protein disclosed herein, the composition disclosed herein, or the pharmaceutical composition disclosed herein. In some embodiments, the disease or disorder is an413165576vlAttorney Docket No. 2019802-0011 inflammatory disease or an autoimmune disease. In some embodiments, the inflammatory disease or an autoimmune disease is type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematous, lupus nephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft versus host disease, psoriasis, alopecia areata, HCV-induced vasculitis, Sjogren’s syndrome, Pemphigus, Ankylosing Spondylitis, Behcet's Disease, Wegener's Granulomatosis, Takayasu's Disease, Autoimmune Hepatitis, Sclerosing Cholangitis, Gougerot- sjogren, chronic obstructive pulmonary disease (COPD) and Macrophage Activation Syndrome, inflammatory bowel disease, or any combination thereof.

[0025] In some embodiments, the inflammatory disease or an autoimmune disease is inflammatory bowel disease, Crohn's disease and ulcerative colitis. In some embodiments, the inflammatory disease or an autoimmune disease is COPD.

[0026] In some embodiments, the present disclosure provides a method of treating a disease or disorder for a subject in need thereof, by administering an ILIO fusion protein comprising (a) means for specifically binding human IL-10 receptor; and (b) an IL10 polypeptide comprising a spacer, an Fc polypeptide and a linker operably linked to the 1L10 polypeptide and to the Fc polypeptide, wherein said IL10 fusion protein has increased myeloid selectivity as compared to an IL10 fusion protein without said spacer, as determined using an in vitro STAT3 phosphorylation assay.

[0027] In some embodiments, the present disclosure provides a method of treating a disease or disorder for a subject in need thereof, by administering an IL10 fusion protein comprising (a) means for specifically binding human IL-10 receptor; and (b) an IL10 polypeptide comprising a spacer, an Fc polypeptide and a linker operably linked to the IL10 polypeptide and to the Fc polypeptide, wherein said IL10 fusion protein has increased myeloid selectivity as compared to an IL10 fusion protein without said spacer, as determined using an in vitro IFNy induction assay.

[0028] In some embodiments, the C-terminus of the Fc polypeptide is linked to the N- terminus of the linker and wherein the N-terminus of the IL10 polypeptide is linked to the C- terminus of the linker. In other embodiments, the disease or disorder is an inflammatory disease or an autoimmune disease. In certain embodiments, the inflammatory disease or an autoimmune513165576vlAttorney Docket No. 2019802-0011 disease is type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematous, lupus nephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft versus host disease, psoriasis, alopecia areata, HCV- induced vasculitis, Sjogren’s syndrome, Pemphigus, Ankylosing Spondylitis, Behcet's Disease, Wegener's Granulomatosis, Takayasu's Disease, Autoimmune Hepatitis, Sclerosing Cholangitis, Gougerot- sjogren, chronic obstructive pulmonary disease (COPD) and Macrophage Activation Syndrome, inflammatory bowel disease, or any combination thereof.

[0029] In some embodiments, the inflammatory disease or an autoimmune disease is inflammatory bowel disease, Crohn's disease and ulcerative colitis. In some embodiments, the inflammatory disease or an autoimmune disease is COPD.

[0030] In some embodiments, the present disclosure provides the use of the IL10 fusion protein, the composition or the pharmaceutical composition, for the treatment of a disease or disorder.

[0031] In some embodiments, the present disclosure provides the use of the IL 10 fusion protein, the composition or the pharmaceutical composition, for the manufacture for the treatment of a disease or disorder.

[0032] BRIEF DESCRIPTION OF THE DRAWING

[0033] FIG. 1 depicts an illustration of a structure of an IL 10 monomeric Fc fusion protein.

[0034] FIG. 2 depicts an enlarged view comparing a portion of the amino acid sequence of an IL 10 dimer with a portion of the amino acid sequence of an IL 10 monomer (having a 6- amino acid spacer insertion sequence).

[0035] FIG. 3A graphically depicts the pSTAT3 mean fluorescence intensity (MFI) as a function of Fusion protein 2 concentration (pM) in the presence of monocytes, T cells or B cells. FIG. 3B graphically depicts the pSTAT3 mean fluorescence intensity (MFI) as a function of Fusion protein 1 concentration (pM) in the presence of monocytes, T cells or B cells.

[0036] FIG. 4A graphically depicts the IFNy induction (as fold change over medium) as a function of rhIL-10 concentration in human T cells. FIG. 4B graphically depicts the fFNy613165576vlAttorney Docket No. 2019802-0011 induction (as fold change over medium) as a function of Fusion protein 2 concentration in human T cells. FIG. 4C graphically depicts the IFNy induction (as fold change over medium) as a function of Fusion protein 1 concentration in human T cells.

[0037] FIG. 5 graphically depicts the percent inhibition of LPS-induced TNF as a function of increasing concentration of Fusion protein 1 or Fusion protein 2.

[0038] FIG. 6 graphically depicts the distal colon histology score in T cell transfer colitis as a function of Fusion protein 1 concentration (mg / kg) or Fusion protein 2 concentration (mg / kg) in comparison to controls (i.e., vehicle and naive).

[0039] FIG. 7 graphically depicts the fFNy mean fluorescence intensity (MFT) of splenic CD4+ Thl cells as a function of Fusion protein 1 concentration (mg / kg) or Fusion protein 2 concentration (mg / kg) in comparison to controls (i.e., vehicle and naive).

[0040] FIG. 8 graphically depicts the platelet numbers (E3 / pL) as a function of Fusion protein 1 concentration (mg / kg) or Fusion protein 2 concentration (mg / kg) in comparison to controls (i.e., "vehicle" and "naive").DETAILED DESCRIPTION

[0041] The present disclosure is described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the disclosure are shown. Indeed, the present disclosures can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that the present disclosure will satisfy applicable legal requirements.

[0042] Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed herein and that modifications and other embodiments are intended to be included within the scope of the appended claims.Overview

[0043] Various strategies, compositions, methods, and kits are provided which can be employed to modulate the immune system.713165576vlAttorney Docket No. 2019802-0011

[0044] In some embodiments, the present disclosure provides an TL10 fusion protein comprising an amino acid sequence at least about 99% identical to SEQ ID NO: 1. In some embodiments, the present disclosure provides an IL 10 fusion protein comprising the amino acid sequence as set forth in SEQ ID NO: 1.

[0045] In some embodiments, the present disclosure provides an IL10 fusion protein consisting of (a) a first polypeptide consisting of an amino acid sequence at least about 99% identical to SEQ ID NO: 1 and (b) a second polypeptide consisting of an amino acid sequence at least about 99% identical to SEQ ID NO: 1, wherein the first polypeptide and the second polypeptide each comprise an Fc region, and wherein the first polypeptide and the second polypeptide are associated with each other by at least one covalent disulfide bond and / or at least one non-covalent interaction between the Fc region of the first polypeptide and the Fc region of the second polypeptide.

[0046] In some embodiments, the present disclosure provides an IL10 fusion protein consisting of (a) a first polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 1 and (b) a second polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 1, wherein the first polypeptide and the second polypeptide each comprise an Fc region, and wherein the first polypeptide and the second polypeptide are associated with each other by at least one covalent disulfide bond and / or at least one non-covalent interaction between the Fc region of the first polypeptide and the Fc region of the second polypeptide.

[0047] In some embodiments, compositions disclosed herein include an IL10 fusion protein comprising: (a) an IL10 polypeptide comprising an amino acid sequence at least about 95% identical to SEQ ID NO: 2; (b) an Fc polypeptide comprising an amino acid sequence at least about 95% identical to SEQ ID NO: 3; and (c) a linker (e.g., a peptide linker) operably located between the IL 10 polypeptide and the Fc polypeptide. Optionally, the C-terminus of the Fc polypeptide is linked to the N-terminus of a linker and the N-terminus of the IL 10 polypeptide is linked to the C-terminus of a linker. Optionally, a linker is a peptide linker that is 14 amino acids, 15 amino acids, or 16 amino acids in length.

[0048] In some embodiments, the present disclosure provides an IL 10 fusion protein comprising: (a) an IL10 polypeptide comprising an amino acid sequence at least about 95% identical to SEQ ID NO: 2; (b) an Fc polypeptide comprising an amino acid sequence at least813165576vlAttorney Docket No. 2019802-0011 about 95% identical to SEQ ID NO: 3; and (c) a linker operably linked to the TL10 polypeptide and to the Fc polypeptide, wherein the C-terminus of the Fc polypeptide is linked to the N- terminus of a linker and wherein the N-terminus of the IL 10 polypeptide is linked to the C- terminus of a linker.

[0049] The present disclosure also provides for a nucleic acid sequence encoding an IL 10 fusion protein as described herein. The present disclosure also provides for a vector comprising a nucleic acid sequence encoding an IL 10 fusion protein as described herein. The present disclosure also provides for a host cell comprising a nucleic acid sequence as described herein. The present disclosure also provides for a vector comprising said nucleic acid sequence encoding an TL10 fusion protein as described herein. The present disclosure also provides for a composition comprising an IL10 fusion protein as described herein. The present disclosure is also directed to methods of making an IL10 fusion protein as described herein. The present disclosure is further directed to methods of using an IL10 fusion protein as described herein or a composition comprising an IL10 fusion protein as described herein. The present disclosure is further directed to kits comprising an IL 10 fusion protein as described herein.Definitions

[0050] Unless otherwise specified, each of the following terms have the meaning set forth in this section.

[0051] Articles: It is to be noted that the term a" or "an" entity refers to at least one of that entity; for example, "a nucleic acid sequence," is understood to represent one or more nucleic acid sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0052] And / or: The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Similarly,913165576vlAttorney Docket No. 2019802-0011 the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

[0053] About or Approximately: As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms "about" or "approximately" when preceding a numerical value indicates the value plus or minus a range of 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% In addition, when the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth (e g., "about 10-20" means "about 10 to about 20").

[0054] Administration: As used herein, the term "administration" or "administering" refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Different routes of administration for an IL 10 fusion protein described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. Moreover, in some embodiments, an IL 10 fusion protein described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.1013165576vlAttorney Docket No. 2019802-0011

[0055] Amino acid: The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, pyroglutamate (referred to herein interchangeably as "pyroGlu" and / or "pGlu") and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones but retain the same basic chemical structure as a naturally occurring amino acid. An alpha-carbon refers to the first carbon atom that attaches to a functional group, such as a carbonyl. A beta-carbon refers to the second carbon atom linked to the alpha-carbon, and the system continues naming the carbons in alphabetical order with Greek letters. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0056] Associated: As used herein the term "associated with" refers to a covalent or non- covalent bond formed between a first amino acid chain and a second amino acid chain. In some embodiments, the term "associated with" means a covalent, non-peptide bond or a non-covalent bond. This association can be indicated by a colon, i.e., (:). In some embodiments, it means a covalent bond except a peptide bond. For example, the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CHI and CL regions are associated by a disulfide bond, and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

[0057] Biologically active: As used herein, refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.1113165576vlAttorney Docket No. 2019802-0011

[0058] Combination therapy: As used herein, the term "combination therapy" refers to a clinical intervention in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g. two or more therapeutic agents or modalities). In some embodiments, the two or more therapeutic regimens may be administered simultaneously. In some embodiments, the two or more therapeutic regimens may be administered sequentially (e.g., a first regimen administered prior to administration of any dose or episode of a second regimen). In some embodiments, two or more therapeutic regimens are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administration of one or more therapeutic agents or modalities to a subject receiving the other agent(s) or modality. In some embodiments, combination therapy does not necessarily require that individual agents be administered together in a single composition (or even necessarily at the same time). In some embodiments, two or more therapeutic agents or modalities of a combination therapy are administered to a subject separately, e.g., in separate compositions, via separate administration routes (e.g., one agent orally and another agent intravenously), and / or at different time points. In some embodiments, two or more therapeutic agents may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity), via the same administration route, and / or at the same time.

[0059] Comparable: As used herein, the term "comparable" refers to two or more agents (e.g., entities or set(s) of conditions), situations, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different1213165576vlAttorney Docket No. 2019802-0011 sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0060] Comprise: Throughout the present disclosure, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of . Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of' are also provided.

[0061] Conservative: "Conservative amino acid substitutions" refer to substitutions of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted nonessential amino acid residue in IL 10 fusion proteins is replaced with another amino acid residue from the same side chain family. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)), the contents of each of which are hereby incorporated by reference herein in their entireties.1313165576vlAttorney Docket No. 2019802-0011

[0062] Expression: The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide.

[0063] Fc ligand: The terms "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" are used interchangeably and refer to the C-terminal region of at least one (preferably two) heavy chain(s) of an antibody. Thus, an Fc region comprises a constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CHI or CL). In IgG, IgA and IgD antibody isotypes, an Fc region comprises two identical protein fragments, derived from a second (CH2) and third (CH3) constant domains of an antibody's two heavy chains; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. An IgG isotype is divided in subclasses in certain species: TgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice. For IgG, an Fc region comprises immunoglobulin domains CH2 and CH3 and the hinge between CHI and CH2 domains. Although the definition of the boundaries of an Fc region of an immunoglobulin heavy chain might vary, as defined herein, a human IgG heavy chain Fc region is defined to stretch from an amino acid residue D221 for IgGl, V222 for IgG2, L221 for IgG3 and P224 for IgG4 to the carboxy-terminus of the heavy chain, wherein the numbering is according to the EU index as in Kabat. A CH2 domain of a human IgG Fc region extends from amino acid 237 to amino acid 340, and a CH3 domain is positioned on C-terminal side of a CH2 domain in an Fc region, i.e., it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of an IgG. As used herein, an Fc region can be a native sequence Fc, including any allotypic variant, or a variant Fc (e g., a non-naturally occurring Fc).

[0064] Fragment: A "fragment" of a material or entity as described herein has a structure that includes a discrete portion of the whole but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises at least1413165576vlAttorney Docket No. 2019802-0011 about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer. In some embodiments, a polymer fragment consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer. The whole material or entity may in some embodiments be referred to as the "parent" of the fragment.

[0065] Fusion: A "fusion" or "fusion protein" as used herein comprises a first amino acid sequence (e.g., a first polypeptide) linked to a second amino acid sequence (e.g., a second polypeptide) with which it is not naturally linked in nature. Amino acid sequences which normally exist in separate proteins can be brought together in a fusion protein, or amino acid sequences which normally exist in a same protein can be placed in a new arrangement in a fusion protein. A fusion protein is created, for example, by chemical synthesis, or by creating and translating a polynucleotide sequence(s) in which the peptide regions are encoded in the desired relationship. A fusion protein can further comprise a second amino acid sequence (e.g., a second polypeptide) associated with the first amino acid sequence (e.g., a first polypeptide) by a covalent, non-peptide bond or a non-covalent bond. Upon transcription / translation, a single protein is made. In this way, multiple proteins, or fragments thereof can be incorporated into a single polypeptide. The term "operably linked" or "operably link" refers to a juxtaposition, with or without a spacer or linker, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to function in an intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function. For example, an IL10 molecule (or variant thereof) may be operably linked to a constant region (e.g., an Fc region or a variant thereof) so as to provide for a stable product with IL 10 receptor-binding activity. The term may also be used with respect to polynucleotides. For example, when a polynucleotide sequence encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc.), it is intended to mean that the resulting polynucleotide-regulatory sequence is linked in such a way that permits regulated expression of the polypeptide from the polynucleotide sequence. Further, e.g., an operable linkage between a1513165576vlAttorney Docket No. 2019802-0011 first polypeptide and a second polypeptide fuses the first and second polypeptides together in frame to produce a single polypeptide fusion protein. In some embodiments, a fusion protein of the present disclosure further comprises a third polypeptide which, as discussed in further detail below, can comprise a linker sequence (e.g., a peptide linker sequence).

[0066] Heterologous: The terms "heterologous" and "heterologous moiety" in reference to a polypeptide or polynucleotide refers to a polypeptide or polynucleotide that originates from a different protein or polynucleotide. In some embodiments, additional components of an IL 10 fusion protein as described herein can originate from the same organism as the other polypeptide components of the fusion protein, or the additional components of an IL 10 fusion protein as described herein can be from a different organism than the other polypeptide components of the fusion protein. For instance, a heterologous polypeptide can be synthetic, or derived from a different species, different cell type of an individual, or the same or different type of cell of distinct individuals. In some embodiments, a heterologous moiety is a polypeptide fused to another polypeptide to produce a fusion polypeptide or fusion protein. In some embodiments, a heterologous moiety is a non-polypeptide moiety such as PEG conjugated to a polypeptide or protein. Non-limiting examples of heterologous moieties disclosed herein are glycine / serine linkers.

[0067] Host cell: The term "in vitro host cell" (or simply "host cell"), as used herein, is intended to refer to a cell that comprises a nucleic acid that is not naturally present in the cell, and can be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny cannot, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Exemplary host cells include, but are not limited to, prokaryotic cells (e.g., E. coli eukaryotic cells, for example, fungal cells (e.g., yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomyces pombe), or various animal cells, such as insect cells (e.g., Sf- 9) or mammalian cells (e.g., HEK293F, e.g., CHO, e.g., COS- 7, e.g., NIH-3T3).

[0068] "Improved", "increased" or "reduced": As used herein, these terms, or grammatically comparable comparative terms, indicate values that are relative to a comparable1613165576vlAttorney Docket No. 2019802-0011 reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be "improved" relative to that obtained with a comparable reference agent. In some embodiments, an assessed value achieved in a subject or system of interest may be "improved" relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance.

[0069] Isolated polynucleotide or isolated nucleic acid molecule. As used herein, an"isolated" polynucleotide or nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source (e.g., in a mouse or a human) of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the language "substantially free" includes preparations of polynucleotide or nucleic acid molecule having less than about 15%, less than about 10%, less than about 5%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% (in particular less than about 10%) of other material, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors and / or other chemicals. In a specific embodiment, a nucleic acid molecule(s) encoding an IL 10 fusion protein described herein is isolated or purified.

[0070] Interleukin-10 (IL-10): As used herein, "Interleukin-10", "IL10", or "IL-10" refers to any native or recombinant IL 10 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and domesticated or agricultural mammals unless otherwise indicated. The term encompasses unprocessed IL10, as well as, any form of IL10 that results from processing in the cell (i.e., a mature form of IL10). The term also encompasses naturally occurring variants and fragments of IL 10, e.g., splice variants or allelic1713165576vlAttorney Docket No. 2019802-0011 variants, and non-naturally occurring variants that have IL10 activity of the naturally occurring IL10. Biologically active fragments and variants of IL10 retain IL10 activity. The phrase "biological activity of IL10" or "IL10 activity" refers to one or more of the biological activities of IL 10, including but not limited to, the ability to stimulate IL 10 receptor-bearing cells and / or the ability to inhibit one or more inflammatory cytokine pathways at the cellular level. Such activity can be measured both in vitro and in vivo.

[0071] Linker: as used herein, a "linker" is used to refer to that portion of a multielement agent that connects different elements to one another. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by a linker. In some embodiments, a polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) are known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1 121-1123), the contents of each of which are hereby incorporated by reference herein in their entireties.

[0072] Linked and fused: The terms "linked" and "fused" as used herein refers to a first amino acid sequence or a first nucleotide sequence covalently or non-covalently joined to a second amino acid sequence or second nucleotide sequence, respectively. A first amino acid or first nucleotide sequence can be directly joined or juxtaposed to a second amino acid or second nucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. In some embodiments, a first amino acid sequence is linked to a second amino acid sequence by a peptide bond or a linker. In some embodiments, a first nucleotide sequence can be linked to a second nucleotide sequence by a phosphodiester bond or a linker. A linker can be a peptide or a polypeptide (for polypeptide chains) or a nucleotide or a1813165576vlAttorney Docket No. 2019802-0011 nucleotide chain (for polynucleotide chains) or any chemical moiety (for both polypeptide and polynucleotide chains). In some embodiments, the term "linked" is indicated by a hyphen (-).

[0073] Naturally occurring: The term "naturally occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature, and which has not been intentionally modified in the laboratory is naturally occurring. The term "native IL 10", also used interchangeably with the term "wild-type IL10", refers to a naturally occurring IL 10

[0074] Nucleic Acid: The terms "nucleic acid sequence" and "nucleic acid molecule" or “polynucleotide” as used herein, are intended to include DNA molecules and RNA molecules. For example, use of the term "polynucleotide" is not intended to limit the present disclosure to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides, can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. A nucleic acid molecule can be single-stranded or double-stranded and can be cDNA. The terms "polynucleotide" and "nucleic acid sequence", which encodes a gene product, e.g., a polypeptide, and are used interchangeably, can include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

[0075] Percent Identity: The terms "percent sequence identity", "% sequence identity", "percent identity", "% identity", "sequence identity", and "identity" are used interchangeably and refer to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids). In some embodiments, polymeric molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical. Calculation of percent1913165576vlAttorney Docket No. 2019802-0011 identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of length of a reference sequence; residues at corresponding positions are then compared. A matched position is any position where an identical amino acid (or nucleic acid) residue is presented in both the candidate and reference sequence. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, e.g., using a mathematical algorithm, as described in the non-limiting examples below. Percent sequence identity may be calculated by determining the number of positions at which the identical amino acid (or nucleic acid) residues occur in both the candidate and reference sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences (i.e., a candidate sequence and a reference sequence) may be accomplished using publicly available software. Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One exemplary suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other exemplary suitable programs include, e.g., ClustalW2, as well as programs including Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa. Amino acid (or nucleic acid) sequences described herein can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10, the contents of which are hereby incorporated by reference herein in their entireties. BLAST nucleotide searches can2013165576vlAttorney Docket No. 2019802-0011 be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, the contents of which are incorporated by reference herein in their entirety. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0076] Pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in a particular form (e.g., in a solid form or a liquid form), and / or may be specifically adapted for, for example: oral administration (for example, as a drenche [aqueous or non-aqueous solutions or suspensions], tablet, capsule, bolus, powder, granule, paste, etc, which may be formulated specifically for example for buccal, sublingual, or systemic absorption); parenteral administration (for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained- release formulation, etc); topical application (for example, as a cream, ointment, patch or spray applied for example to skin, lungs, or oral cavity); intravaginal or intrarectal administration (for example, as a pessary, suppository, cream, or foam); ocular administration; nasal or pulmonary administration, etc. In some embodiments, a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Except insofar as any conventional media or agent is incompatible with the IL 10 fusion proteins, use thereof in the compositions is contemplated.

[0077] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as2113165576vlAttorney Docket No. 2019802-0011 disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0078] Polypeptide, Protein, Peptide: The terms "polypeptide, " "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, or an assembly of multiple polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. A "fusion protein", as used herein, refers to a protein comprising one or more polypeptides that are associated to perform certain functions.

[0079] Spacer: The term "spacer" or “spacer insertion sequence” as used herein refers to an artificial amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more amino acid residues, or a length of between about 5 and about10, about 12, about 15, about 18, about 20, about 22, about 25, about 28, about 30, about 32, about 35, about 38, about 40, about 42, about 45, about 48, about 50, about 55, about 60, about65, about 70, about 75, about 80, about 85, about 90, about 95, about 100 or more amino acid residues. In some embodiments, a spacer is joined by peptide bonds. In some embodiments, a spacer is used to link one or more polypeptides. A spacer may or may not have a secondary structure. Spacer sequences are known in the art, see, for example, Holliger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993); Poljak et al., Structure 2: 1121-1123 (1994), the contents of each of which are incorporated by reference herein in their entireties. Any suitable spacers known in the art can be used.

[0080] Specific binding: The term "specific binding", "specifically binding" or "specifically binds" as used herein refers to a non-random binding reaction between two molecules, such as for example between a fusion protein (e.g., an IL10 fusion protein) as described herein and a receptor (e.g., IL10 receptor). In some embodiments, the fusion proteins provided herein specifically bind IL 10 receptor with a binding affinity (KD) of < 10'6M (e.g., < 5xl0‘7M, < 2xl0‘7M, < IO’7M, < 5xl0’8M, < 2xl0’8M, < 10'8M, < 5xl0’9M, < 2xl0’9M, < 10’9M, or < 10’10M). KD as used herein refers to the ratio of the dissociation rate to the2213165576vlAttorney Docket No. 2019802-0011 association rate (KOFF / KON), which as will be appreciated by one of ordinary skill in the art, may be determined using methods known in the art, including, but not limited to, surface plasmon resonance methods, for example, using an instrument such as Biacore.

[0081] Subject: As used herein, the terms "subject" and "patient" refer to an organism, such as a human, that receives treatment for a particular disease or condition as described herein. For instance, a patient, such as a human patient, may receive treatment of an inflammatory or autoimmune disease to simultaneously inhibit inflammation and promote tolerance (e.g., to promote the induction of tolerogenic myeloid cells).

[0082] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0083] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an agent (e.g., an entity or condition) that, when administered to a subject, has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a "therapeutic agent" is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a "therapeutic agent" is an agent for which a medical prescription is required for administration to humans.

[0084] Therapeutic dose: A "therapeutically effective amount," "therapeutic dose," "dose," "effective dose," "effective dosage," or "dosing amount" as used (interchangeably) herein, means a dose that achieves a therapeutic goal, as described herein. In some embodiments, a "therapeutically effective amount" of an IL 10 fusion protein refers to the amount of an IL 10 fusion protein sufficient to elicit a desired biological response. As will be appreciated by one of ordinary skill in the art, the absolute amount of a particular IL 10 fusion protein that is effective can vary depending on such factors as the desired biological endpoint, an IL 10 fusion protein to2313165576vlAttorney Docket No. 2019802-0011 be delivered, the target cell or tissue, and the like. An effective amount can be administered in a single dose or can be achieved by administration of multiple doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses). The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0085] Treatment: The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on, or administering an active agent to, a subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease or enhancing overall survival. Treatment can be of a subject having a disease or a subject who does not have a disease (e.g., for prophylaxis). When provided prophylactically, an IL10 fusion protein as disclosed herein is provided in advance of any symptom. The prophylactic administration of an IL10 fusion protein serves to prevent or attenuate any subsequent symptoms.

[0086] Variant: Also included in the present disclosure are fragments or variants of polypeptides, and any combination thereof. The term "fragment" or "variant" when referring to a polypeptide of the present disclosure includes any polypeptide which retains at least some property (e.g., IL10 binding activity for IL10R) of the reference polypeptide. Fragments of a polypeptide of the present disclosure includes proteolytic fragments, as well as deletion fragments, but do not include the naturally occurring full-length polypeptide (or mature polypeptide). Variants of a polypeptide of the present disclosure include fragments as described above, and also a polypeptide(s) with an altered amino acid sequence due to amino acid modifications (e.g., substitution mutations, deletion mutations, insertion mutations, or any combination thereof). Variants of a polypeptide of the present disclosure can be naturally or non- naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variants of a polypeptide of the present disclosure can comprise conservative or non-conservative amino acid substitutions, deletions or insertions. In some embodiments, polypeptide variants include, e.g., modified polypeptides relative to a natural (or wildtype) polypeptide. Modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety,2413165576vlAttorney Docket No. 2019802-0011 covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:210-19 (2010)), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0087] Vector: The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors") In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, also included are other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0088] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.

[0089] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, in some embodiments, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and2513165576vlAttorney Docket No. 2019802-0011Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0090] Various embodiments are described in further detail herein.Interleukin-10 Fusion Proteins

[0091] Disclosed herein is an IL10 fusion protein comprising at least three components: (a) a first polypeptide comprising an Interleukin- 10 (IL10) polypeptide; (b) a second polypeptide comprising an Fc polypeptide; and (c) a linker (e.g., a peptide linker) operably linked between the first polypeptide and the second polypeptide. In some embodiments, an IL10 fusion protein as described herein is more myeloid selective than a reference protein. In some embodiments, a reference protein comprises or is a wildtype IL10 polypeptide consisting of SEQ ID NO: 6. In some embodiments, a reference protein comprises or is an IL10 fusion protein consisting of SEQ ID NO: 8.Interleukin-10

[0092] In some embodiments, an IL10 fusion protein as described herein comprises a first polypeptide comprising an IL10 polypeptide fused to a second polypeptide comprising an Fc polypeptide. In some embodiments, an IL 10 fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, an IL10 fusion protein comprises a first polypeptide consisting of SEQ ID NO: 2. In some embodiments, a first polypeptide comprises an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% (e.g., 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%), or 100% identical to SEQ ID NO:2. In some embodiments, a first polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO:2.

[0093] In some embodiments, an IL 10 polypeptide is a recombinant IL10 polypeptide from a mammal, such as a primate (e.g., a human). For example, in some embodiments, a2613165576vlAttorney Docket No. 2019802-0011 recombinant IL10 polypeptide is a monomeric IL10 polypeptide (e.g., a wildtype IL10 comprising a 6-amino acid spacer insertion sequence). An exemplary monomeric IL10 polypeptide (with a 6-amino acid spacer insertion sequence underlined) is shown below as SEQ ID NO: 2.

[0094] In some embodiments, a spacer insertion sequence can be a 1 -amino acid sequence. In some embodiments, a spacer insertion sequence can be a 2-amino acid sequence. In some embodiments, a spacer insertion sequence can be a 3 -amino acid sequence. In some embodiments, a spacer insertion sequence can be a 4-amino acid sequence. In some embodiments, a spacer insertion sequence can be a 5-amino acid sequence. In some embodiments, a spacer insertion sequence can be a 6-amino acid sequence. In some embodiments, a spacer insertion sequence can be a 7-amino acid sequence. In some embodiments, a spacer insertion sequence can be an 8-amino acid sequence. In some embodiments, a spacer insertion sequence can be a 9-amino acid sequence. In some embodiments, a spacer insertion sequence can be a 10-amino acid sequence.

[0095] In some embodiments, a spacer insertion sequence comprises glycine amino acid residues. In some embodiments, a spacer insertion sequence comprises a combination of glycine and serine amino acid residues, e g., GGGSGG (SEQ ID NO: 5).

[0096] In some embodiments, a spacer insertion sequence is inserted between helices D and E of the IL 10 polypeptide as shown in FIG. 2. In some embodiments, a spacer insertion sequence is inserted between amino acids N116 and KI 17 of the IL10 polypeptide as shown in FIG. 2

[0097] In some embodiments, IL 10 polypeptides described herein are expressed as a fusion protein. IL 10 fusion proteins as described herein specifically bind human IL10R (hILlOR). In some embodiments, a IL 10 fusion protein described herein binds to human IL10R with high affinity. In some embodiments, a IL 10 fusion protein described herein binds to human IL10R with a KD of about 0.1 pM to about 1 pM as determined by surface plasmon resonance2713165576vlAttorney Docket No. 2019802-0011(SPR) assay. In some embodiments, an IL10 fusion protein described herein binds to human IL10R with a KD of about 0.1 pM, about 0.2 pM, about 0.5 pM, about 1 pM, about 2 pM, about 5 pM, about 10 pM, about 20 pM, about 50 pM, about 100 pM, about 200 pM, about 400 pM, about 600 pM, about 800 pM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 8 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 800 nM, about 1 pM, about 2 pM, about 5 pM, about 10 pM, about 20 pM, about 50 pM, about 100 pM, about 200 pM, about 500 pM, or about 1 mM or more as determined by surface plasmon resonance (SPR) assay.

[0098] In some embodiments, an IL 10 fusion protein described herein binds to human IL10R with a KD of about 100 nM or less, about 95 nM or less, about 90 nM or less, about 85 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 65 nM or less, about 60 nM or less, about 55 nM or less, about 50 nM or less, about 45 nM or less, about 40 nM or less, about 35 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 18 nM or less, about 16 nM or less, about 14 nM or less, about 12 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1.5 nM or less, about 1 nM or less, about 0.8 nM or less, about 0.6 nM or less, about 0.4 nM or less, or about 0.2 nM or less as determined by SPR.

[0099] In some embodiments, an IL 10 fusion protein described herein binds to human IL10R with a KD of about 1 pM or more, about 2 pM or more, about 3 pM or more, about 4 pM or more, about 5 pM or more, about 6 pM or more, about 7 pM or more, about 8 pM or more, about 9 pM or more, about 10 pM or more, about 15 pM or more, about 20 pM or more, about 25 pM or more, about 30 pM or more, about 40 pM or more, about 50 pM or more, about 60 pM or more, about 70 pM or more, about 80 pM or more, about 90 pM or more, about 100 pM or more, about 150 pM or more, about 200 pM or more, about 250 pM or more, about 300 pM or more, about 400 pM or more, about 500 pM or more, about 600 pM or more, about 700 pM or more, about 800 pM or more, about 900 pM or more, or about 1 mM or more as determined by SPR.2813165576vlAttorney Docket No. 2019802-0011

[0100] In some embodiments, an IL10 fusion protein described herein binds to human IL10R with a Koof about 1 nM. In some embodiments, an IL10 fusion protein binds to human IL10R with a Koof about 2 nM. In some embodiments, an IL10 fusion protein described herein binds to human IL10R with a KOFF of about 1 x I O3s1to about P I O3s ' as determined by SPR. In some embodiments, an IL 10 fusion protein binds to human IL10R with a KOFF of 1 x 102s1to P I O3s ', IxlO-3s ' to P I O4s ', IxlO-5s ' to PIO-6s ', P IO6s ' to P IO-7s ' or 1 107s1to 1 x 10xs1as measured by SPR.

[0101] As described herein, IL 10 encompasses unprocessed IL 10. In some embodiments, IL10 encompasses any form of IL10 that results from processing in a cell (i.e., a mature form of IL10). In some embodiments, TL10 encompasses naturally occurring variants and fragments of IL10, e.g., splice variants or allelic variants, as well as non-naturally occurring variants.

[0102] An amino acid sequence of an exemplary mature form of human IL10 (without the 18-amino acid signal sequence) is shown in SEQ ID NO: 6 below.

[0103] An amino acid sequence of an exemplary unprocessed form of human IL 10 (with the 18-amino acid signal sequence) is shown in SEQ ID NO: 7 below.

[0104] Biologically active fragments and variants of IL10 are also provided. Such IL10 active fragments or variants are fragments or variants of IL 10 that retain IL 10 activity. In some embodiments, biological activity of IL10 refers to the ability to stimulate IL10 receptor bearing cells. Such activity can be measured both in vitro and in vivo.

[0105] Biologically active fragments and variants of IL10 can be employed in the IL10 fusion proteins disclosed herein. In some embodiments, such a biologically active fragment can2913165576vlAttorney Docket No. 2019802-001 1 comprise at least 30, at least 31 , at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, at least 445, at least 450, at least 455, at least 460, at least 465, at least 470, at least 475, at least 480, at least 485, at least 490, at least 495, at least 500 or more continuous amino acids of SEQ ID NO: 2.

[0106] In some embodiments, a biologically active variant of IL10 can comprise at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more (e.g., 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0107] In some embodiments, a biologically active variant of IL 10 comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a biologically active variant of IL10 comprises at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a biologically active variant of IL 10 comprises at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a biologically active variant of IL10 comprises at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a biologically active variant of IL10 comprises at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a biologically active variant of IL 10 comprises 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a biologically active variant of IL10 comprises the amino acid3013165576vlAttorney Docket No. 2019802-0011 sequence set forth in SEQ ID NO: 2. Tn some embodiments, a biologically active variant of IL10 consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0108] Polynucleotide fragments and variants encoding biologically active IL 10 polypeptides are further provided. In some embodiments, such polynucleotide fragments and variants can comprise at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500, at least 525, at least 550, at least 575, at least 600, at least 625, at least 650, at least 675, at least 700, at least 725, at least 750, at least 775, at least 800, at least 825, at least 850, at least 875, at least 900, at least 925, at least 950, at least 975, at least 1000, at least 1025, at least 1050, at least 1075, at least 1 100, at least 1125, at least 1150, at least 1175, at least 1200, at least 1203, at least 1206, at least 1209, at least 1212, at least 1215, or more continuous nucleotides of polypeptide encoding SEQ ID NO: 2, and continue to encode a biologically active IL 10 polypeptide.

[0109] In some embodiments, a polynucleotide variant or fragment can comprise at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more (e.g., 90. 1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%; 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%;92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%; 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%; 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%; 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%;96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%; 97.1%, 97.2%, 97.3%,97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%; 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%,98.7%, 98.8%, 98.9%; and 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2.

[0110] In some embodiments, a polynucleotide variant or fragment can comprise at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a polynucleotide variant or fragment can comprise at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a polynucleotide variant or fragment can comprise at least 97% sequence identity to the amino acid sequence set3113165576vlAttorney Docket No. 2019802-0011 forth in SEQ ID NO: 2. In some embodiments, a polynucleotide variant or fragment can comprise at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a polynucleotide variant or fragment can comprise at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a polynucleotide variant or fragment can comprise 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a polynucleotide variant or fragment comprises the sequence set forth in SEQ ID NO: 2. In some embodiments a polynucleotide variant or fragment consists of the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, such polynucleotide variants or fragments encode a biologically active IL 10 polypeptide.

[0111] Exemplary polypeptide sequences of IL 10 are recited in Table 1 below.Table 1: Exemplary IL10 amino acid sequences

[0112] In some embodiments, IL 10 fusion proteins described herein may also include one or more amino acid modification(s) relative to a wild-type IL 10 polypeptide. In some embodiments, one or more modification(s) is a substitution of an amino acid of IL10 from an alanine to an amino acid such as arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.3213165576vlAttorney Docket No. 2019802-0011

[0113] In some embodiments, one or more modification(s) is a substitution of an amino acid of IL 10 from a threonine to an amino acid such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, or valine.

[0114] In some embodiments, one or more modification(s) is a substitution of an amino acid of IL 10 from a reactive amino acid, e.g., a cysteine, to an amino acid such as alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, one or more modification(s) is a substitution from a cysteine to a serine. In some embodiments, one or more modification(s) is a substitution from a cysteine to an alanine. In some embodiments, one or more modification(s) is a substitution from a cysteine to a valine.Immunoglobulin Constant Region or Portion Thereof

[0115] An 1L10 fusion protein of the present disclosure can further comprise an immunoglobulin constant region (e.g., an Fc region) or portion thereof. Depending on isotype (e.g.,, IgG, IgM, IgA, IgD, or IgE), a constant region can be comprised of three or four CH domains. Some isotype (e.g., IgG) constant regions also contain a hinge region. See Janeway et al. 2001, Immunobiology’, Garland Publishing, N.Y., N.Y., the contents of which are incorporated by reference herein in their entirety.

[0116] An immunoglobulin constant region (e.g., an Fc region) or a portion thereof for producing an IL10 fusion protein of the present disclosure may be obtained from a number of different sources. In some embodiments, an Fc region or a portion thereof is derived from a human immunoglobulin. It is understood, however, that an Fc region or a portion thereof may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g., a mouse, e.g., a rat, e.g., a rabbit, e.g., a guinea pig) or non-human primate (e.g., a chimpanzee, e.g., a macaque) species. Moreover, an Fc region or a portion thereof may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including IgGl, IgG2, IgG3 and IgG4. In some embodiments, an Fc region of an IL10 fusion protein of the present disclosure is the a region of a human IgGl. In3313165576vlAttorney Docket No. 2019802-0011 some embodiments, an Fc region of an IL10 fusion protein of the present disclosure is not an Fc region of an IgA.

[0117] A variety of Fc region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains sequence can be selected having a particular effector function (or lacking a particular effector function) or with a particular modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable 1g constant region sequences (e.g., hinge, CH2, and / or CH3 sequences, or portions thereof) can be derived from these sequences using art recognized techniques. Genetic material obtained using any of the foregoing methods may then be altered or synthesized to obtain polypeptides of the present disclosure. It will further be appreciated that the scope of this disclosure encompasses alleles, variants and mutations of constant region DNA sequences.

[0118] Sequences of an Fc region or a portion thereof can be cloned, e.g., using the polymerase chain reaction and primers which are selected to amplify the domain of interest. To clone a sequence of an Fc region or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymph cells, reverse transcribed into DNA, and antibody genes amplified by PCR. PCR amplification methods are described in detail in U.S. Pat. Nos.4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, e.g., "PCR Protocols: A Guide to Methods and Applications" Innis et al. eds., Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270), the contents of each of which are incorporated by reference herein in their entireties. PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.Numerous primer sets suitable for amplification of antibody genes are known in the art (e.g., 5’ primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160: 1250), the contents of each of which are incorporated by reference herein in their entireties. The cloning of antibody sequences is further described in Newman et al., U.S.3413165576vlAttorney Docket No. 2019802-0011Pat. No. 5,658,570, filed January 25, 1995, the contents of which are incorporated by reference herein in its entirety.

[0119] An Fc region used herein can include all domains and the hinge region or portions thereof. In some embodiments, an Fc region or a portion thereof comprises a CH2 domain, a CH3 domain, and a hinge region, i.e., an Fc region or an FcRn binding partner.

[0120] In some embodiments, an Fc region is derived from a human immunoglobulin. It is understood, however, that an Fc region may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g., a mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. Moreover, the polypeptide of the Fc domains or portions thereof may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including IgGl, IgG2, IgG3 and IgG4. In some embodiments, a human isotype IgGl is used.

[0121] In some embodiments, an IL10 fusion protein as described herein comprises an Fc variant that confers a change in at least one effector function imparted by an Fc region of the corresponding wild-type Fc region (e.g., an improvement or reduction in the ability of the Fc region to bind to Fc receptors (e.g., FcyRI, FcyRII, or FcyRIII) or complement proteins (e g., Clq), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complementdependent cytotoxicity (CDCC)). In some embodiments, the Fc variant provides an engineered cysteine residue.

[0122] An Fc region of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by Fc receptors. Such modifications include modifications remote from the Fc receptor contact sites as well as modifications within the contact sites that preserve or even enhance binding to the Fc receptors. For example, the following single amino acid residues in human IgGl Fc (Fc yl) can be substituted without significant loss of Fc binding affinity for Fc receptors: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A33OQ, P331A, E333A,3513165576vlAttorney Docket No. 2019802-0011K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wild type proline substituted by alanine at position number 238. As an example, a specific embodiment incorporates the N297A mutation, removing a highly conserved N-glycosylation site. In addition to alanine other amino acids may be substituted for the wildtype amino acids at the positions specified above. Mutations may be introduced into Fc giving rise to more than one hundred Fc regions distinct from the native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more Fc regions.Moreover, in some embodiments, one of two heavy chain polypeptide sequences of an Fc region of an IL10 fusion protein of the disclosure may be modified (e.g., mutated) while the other heavy chain polypeptide sequence of an Fc region of an IL 10 fusion protein may not be modified (e.g., mutated) at all, or they both may be modified (e.g., mutated) but with different modification (e.g., mutations).

[0123] Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgGl "ELLG" to the corresponding sequence from IgG2 "PVA" (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRIII, which mediate various effector functions will not bind to IgGl when such mutations have been introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613, the contents of each of which are incorporated by reference herein in their entireties.

[0124] An exemplary Fc polypeptide of the present disclosure is hlgGl ,3f as shown below as SEQ ID NO: 3.3613165576vlAttorney Docket No. 2019802-0011Linker

[0125] An IL10 fusion protein of the present disclosure can further comprise a linker. In some embodiments, a linker can operably link a first polypeptide to a second polypeptide from N-terminus to C-terminus, e.g., N-ILlO-linker-Fc-C. In some embodiments, a linker can operably link a second polypeptide to a first polypeptide from N-terminus to C-terminus, e.g., N- Fc-linker-ILlO-C.

[0126] In some embodiments, an IL 10 fusion protein of the present disclosure comprises a linker sequence located between an IL 10 polypeptide and an Fc polypeptide. A linker can be of any length. For example, a linker can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids. In some embodiments, a linker of the present disclosure has at least one amino acid and less than about 100 amino acids, less than about 90 amino acids, less than about 80 amino acids, less than about 70 amino acids, less than about 60 amino acids, less than about 50 amino acids, less than about40 amino acids, less than about 30 amino acids, less than about 20 amino acids, less than about19 amino acids, less than about 18 amino acids, less than about 17 amino acids, less than about16 amino acids, less than about 15 amino acids, less than about 14 amino acids, less than about13 amino acids, less than about 12 amino acid, less than about 11 amino acids, or less than about 10 amino acids. In some embodiments, a linker sequence comprises glycine amino acid residues. In some embodiments, a linker sequence comprises a combination of glycine and serine amino acid residues.

[0127] In some embodiments, a linker is a peptide linker containing amino acids such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine. Amino acid residues which comprise a peptide linker include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. In some embodiments, linkers suitable for use herein may further include one or more groups including Ci-Ce alkylene, Ci-Ce heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C63713165576vlAttorney Docket No. 2019802-0011 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, or combinations thereof, each of which may be optionally substituted. Non-limiting examples of such groups include (CH2)p, (CH2CH20)p, and -(C=0)(CH2) - units, wherein p is an integer from 1-6, independently selected for each occasion. In some embodiments, suitable linkers may contain groups having solubility enhancing properties. Linkers including the (CH2CH2O) unit (polyethylene glycol, PEG), for example, can enhance solubility, as can alkyl chains substituted with amino, sulfonic acid, phosphonic acid or phosphoric acid residues. Linkers including such moieties are disclosed in, for example, U.S. Patent Nos. 8,236,31 9 and 9,504,756, the contents of each of which are incorporated herein by reference in their entirety as it pertains to linkers suitable for covalent conjugation. In some embodiments, a linker may include one or more of a hydrazine, a disulfide, a thioether, a dipeptide, a p-aminobenzyl (PAB) group, an optionally substituted Ci-Ce alkyl, an optionally substituted Ci-Ce heteroalkyl, an optionally substituted C2- Ce alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2- Ce alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted C3-C6 cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, a solubility enhancing group, acyl, or -(C=0)- group. In some embodiments, a linker includes a p-aminobenzyl group (PAB). In some embodiments, the p- aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit. In some embodiments, the p- aminobenzyl group is part of a p- aminobenzylamido unit. In some embodiments, a linker comprises PAB, Val-Cit-PAB, Vai-Ala- PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)- PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn- PAB, or Ala-PAB. In some embodiments, a linker comprises a combination of one or more of a peptide, oligosaccharide, -(CH2)p-, - (CH2CH20)P-, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe- Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB. In some embodiments, a linker comprises a -(C=0)(CH2) - unit, wherein p is an integer from 1 -6, and wherein (Ac) represents acetyl lysine (or acetylated lysine). In some embodiments, a linker comprises a -(CH2)n- unit, wherein n is an integer from 2 to 6. In some embodiments, a linker can be used to conjugate an Fc region to an IL 10 polypeptide, for example those linkers that are covalently bound to the Fc region on one end of a linker and, on the other end of a linker, contain a chemical moiety formed from a coupling reaction between a reactive substituent present on a linker and a reactive substituent present within the IL10 polypeptide (and vice versa). Reactive3813165576vlAttorney Docket No. 2019802-0011 substituents that may be present within an IL10 polypeptide include, without limitation, hydroxyl moi eties of serine, threonine, and tyrosine residues; amino moi eties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), halohet eroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids. It will be recognized by one of skill in the art that any one or more of the chemical groups, moieties and features disclosed herein may be combined in multiple ways to form linkers useful for conjugation of the Fc regions and IL10 polypeptides as disclosed herein. Further linkers useful in conjunction with the compositions and methods described herein, are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the contents of which are incorporated herein by reference in their entirety.

[0128] In some embodiments, an IL 10 fusion protein comprising an Fc region and an IL10 polypeptide may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent to one another or separated by a region encoding a linker peptide which does not destroy the desired properties of an IL 10 fusion protein. Alternative linkers can be used to so as to increase the resistance of an IL 10 fusion protein to proteolytic degradation (for example, linkers containing D-amino acids), in order to enhance the solubility of an IL 10 fusion protein (for example, hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (for example, a linker containing cysteine residues that form intramolecular or intermol ecular disulfide bonds), or to attenuate the immunogenicity of an IL 10 fusion protein (for example, linkers containing glycosylation sites). It will also be understood by one of ordinary skill in the art that the amino acid sequence of the IL10 polypeptide described herein can be modified such that they vary in amino acid sequence from the IL 10 polypeptide from which they were derived.

[0129] In some embodiments, an IL10 fusion protein of the present disclosure comprises a linker operably linked between the first polypeptide and the second polypeptide. In some embodiments, the fusion protein comprises a glycine / serine linker. Such glycine / serine linkers can comprise any combination of the amino acid residues, including, but not limited to, the peptide GGGSGGGGSGGGGS (SEQ ID NO: 4).3913165576vlAttorney Docket No. 2019802-0011

[0130] In some embodiments, an IL10 fusion protein disclosed herein includes any one or more of the multiple linker amino acid sequences as provided in Table 2.Table 2: Linker amino acid sequencesFusion Protein

[0131] In some embodiments, an IL10 fusion protein of the present disclosure comprises an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% (e.g., at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%), or 100% identical to SEQ ID NO: 1.

[0132] In some embodiments, an IL10 fusion protein of the present disclosure comprises an amino acid sequence at least about 98% identical to SEQ ID NO: 1. In some embodiments, an IL10 fusion protein of the present disclosure comprises an amino acid sequence at least about 99% identical to SEQ ID NO: 1. In some embodiments, an IL10 fusion protein of the present disclosure comprises an amino acid sequence 100% identical to SEQ ID NO: 1. In some embodiments, an IL10 fusion protein of the present disclosure comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, an IL10 fusion protein of the present disclosure consists of the amino acid sequence of SEQ ID NO: 1.

[0133] An exemplary IL10 fusion protein of the present disclosure is shown below as SEQ ID NO: 1.4013165576vlAttorney Docket No. 2019802-0011

[0134] In some embodiments, IL 10 fusion proteins disclosed herein have an extended half-life compared to a wildtype IL10 polypeptide. In some embodiments, IL 10 fusion proteins disclosed herein have an extended half-life compared to a wildtype IL 10 polypeptide consisting of SEQ ID NO: 6.

[0135] In some embodiments, an extended half-life is increased by at least about 1.5- fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8- fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 10.5-fold, at least about 11-fold, at least about 11.5-fold, at least about 12-fold, at least about 12.5-fold, at least about 13-fold, at least about 13.5-fold, at least about 14-fold, at least about 14.5-fold, at least about 15-fold, at least about 15.5-fold, at least about 16-fold, at least about 16.5-fold, at least about 17-fold, at least about 17.5-fold, at least about 18-fold, at least about 18.5-fold, at least about 19-fold, at least about 19.5-fold, at least about 20-fold, at least about 20.5-fold, at least about 21-fold, at least about 21.5-fold, at least about 22-fold, at least about 22.5-fold, at least about 23-fold, at least about 23.5-fold, at least about 24-fold, at least about 24.5-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, or at least about 1000-fold or more compared to the half-life of an IL10 polypeptide consisting of SEQ ID NO: 6.4113165576vlAttorney Docket No. 2019802-0011

[0136] In some embodiments, TL10 fusion proteins disclosed herein exhibit an extended half-life that is no less than about 1.5-fold, no less than about 2.0-fold, no less than about 2.5- fold, no less than about 3.0-fold, no less than about 3.5-fold, no less than about 4.0-fold, no less than about 4.5-fold, no less than about 5.0-fold, no less than about 5.5-fold, no less than about 6.0-fold, no less than about 6.5-fold, no less than about 7.0-fold, no less than about 7.5-fold, no less than about 8.0-fold, no less than about 8.5-fold, no less than about 9.0-fold, no less than about 9.5-fold, no less than about 10.0-fold, no less than about 10.5-fold, no less than about 11.0- fold, no less than about 11.5-fold, no less than about 12.0-fold, no less than about 12.5-fold, no less than about 13.0-fold, no less than about 13.5-fold, no less than about 14.0-fold, no less than about 14.5-fold, no less than about 15.0-fold, no less than about 15.5-fold, no less than about 16.0-fold, no less than about 16.5-fold, no less than about 17.0-fold, no less than about 17.5-fold, no less than about 18.0-fold, no less than about 18.5-fold, no less than about 19.0-fold, no less than about 19.5-fold, no less than about 20.0-fold, no less than about 20.5-fold, no less than about 21.0-fold, no less than about 21.5-fold, no less than about 22.0-fold, no less than about 22.5-fold, no less than about 23.0-fold, no less than about 23.5-fold, no less than about 24.0-fold, no less than about 24.5-fold, and no less than about 25.0-fold compared to the half-life of the IL10 polypeptide consisting of SEQ ID NO: 6.

[0137] In some embodiments, IL10 fusion IL10 fusion proteins disclosed herein have an extended half-life that is no greater than about 25.0-fold, no greater than about 24.5-fold, no greater than about 24.0-fold, no greater than about 23.5-fold, no greater than about 23.0-fold, no greater than about 22.5-fold, no greater than about 22.0-fold, no greater than about 21 5-fold, no greater than about 21.0-fold, no greater than about 20.5-fold, no greater than about 20.0-fold, no greater than about 19.5-fold, no greater than about 19.0-fold, no greater than about 18.5-fold, no greater than about 18.0-fold, no greater than about 17.5-fold, no greater than about 17.0-fold, no greater than about 16.5-fold, no greater than about 16.0-fold, no greater than about 15.5-fold, no greater than about 15.0-fold, no greater than about 14.5-fold, no greater than about 14.0-fold, no greater than about 13.5-fold, no greater than about 13.0-fold, no greater than about 12.5-fold, no greater than about 12.0-fold, no greater than about 11.5-fold, no greater than about 11.0-fold, no greater than about 10.5-fold, no greater than about 10.0-fold, no greater than about 9.5-fold, no greater than about 9.0-fold, no greater than about 8.5-fold, no greater than about 8.0-fold, no greater than about 7.5-fold, no greater than about 7.0-fold, no greater than about 6.5-fold, no4213165576vlAttorney Docket No. 2019802-0011 greater than about 6.0-fold, no greater than about 5.5-fold, no greater than about 5.0-fold, no greater than about 4.5-fold, no greater than about 4.0-fold, no greater than about 3.5-fold, no greater than about 3.0-fold, no greater than about 2.5-fold, no greater than about 2.0-fold, and no greater than about 1.5-fold compared to the half-life of the IL10 polypeptide consisting of SEQID NO: 6.

[0138] In some embodiments, an IL 10 fusion protein of the present disclosure has an improved activity over the wildtype 1L10 polypeptide or the wildtype 1L10 dimer Fc fusion protein. For example, an IL 10 fusion protein is more myeloid selective in comparison to the wildtype IL10 polypeptide consisting of SEQ ID NO: 6 or the IL10 fusion polypeptide consisting of SEQ ID NO: 8 as shown below.

[0139] Described herein, the term "secretory signal sequence" denotes a polynucleotide sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs a larger polypeptide through a secretory pathway of the cell in which it is synthesized. A larger polypeptide is commonly cleaved to remove a secretory peptide during transit through the secretory pathway. As used herein, a "mature" form of a fusion protein or polypeptide comprises a processed form of a polypeptide that has had a secretory peptide removed. As used herein, the "unprocessed" form of the fusion protein retains the secretory peptide sequence.

[0140] Biologically active fragments and variants of a mature or unprocessed form of an IL10 fusion protein, and a polynucleotide encoding the same, are also provided. In some embodiments, a functional polypeptide fragment comprises at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least4313165576vlAttorney Docket No. 2019802-0011 about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 375, at least 400, at least 401, at least 402, at least 403, at least 404, at least 405, or more continuous amino acids of SEQ ID NO: 1.

[0141] In some embodiments, a functional polypeptide variant can comprise at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or greater sequence (e.g., at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%) identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0142] In some embodiments, a functional polypeptide variant comprises at least about 98% identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a functional polypeptide variant comprises at least about 99% identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a functional polypeptide variant comprises 100% identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a functional polypeptide variant comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, a functional polypeptide variant consists of the sequence set forth in SEQ ID NO: 1.

[0143] Polynucleotides encoding biologically active variants and fragments of an IL 10 fusion protein are further provided. In some embodiments, such polynucleotides can comprise at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1150, at least about 1200, at least 1203, at least 1206, at least 1212, at least 1215 continuous nucleotides encoding the polypeptides set forth in SEQ ID NO: 1 and continue to encode a functional IL10 fusion protein.

[0144] It is further recognized that the components of an IL10 fusion protein as disclosed herein can be positioned in various structural orientations. In some embodiments, an IL 10 polypeptide is positioned at the N-terminus and an Fc polypeptide is positioned at the C-terminus of an IL10 fusion protein. In some embodiments, an Fc polypeptide is positioned at the N- terminus and an IL10 polypeptide is positioned at the C-terminus of an IL10 fusion protein.4413165576vlAttorney Docket No. 2019802-0011

[0145] In some embodiments, an IL10 fusion protein forms a dimer. In some embodiments, an IL10 fusion protein is a monomer. In some embodiments, an IL10 fusion protein is a dimer comprising two identical monomers, and monomers are associated with each other only through their Fc regions (via covalent bonds or non-covalent bonds).

[0146] In some embodiments, an IL10 fusion protein is more stable than an IL10 fusion protein set forth in SEQ ID NO: 8. In some embodiments, an IL10 fusion protein has one or more properties including (i) increased thermodynamic stability compared to a reference protein; (ii) increased resistance to degradation compared to a reference protein; (iii) increased resistance to modifications compared to a reference protein; or (iv) increased stability in vivo compared to a reference protein, wherein the reference protein is the wildtype IL10 polypeptide consisting of SEQ ID NO: 6 or the IL10 fusion polypeptide consisting of SEQ ID NO: 8.Polynucleotides

[0147] In some embodiments provided herein are polynucleotides, e.g., DNA or RNA, comprising a nucleic acid sequence encoding an 1L10 fusion protein as described herein, as well as vectors comprising such polynucleotides, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells. In some embodiments, provided herein are polynucleotide sequences that encode polypeptide sequences of SEQ ID NO: 1 and variants thereof.

[0148] Polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Nucleotide sequences encoding fusion proteins described herein, and modified versions of these IL 10 fusion proteins can be determined using methods, e.g., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the fusion protein. Such a polynucleotide encoding an IL 10 fusion protein can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6, the contents of each of which are hereby incorporated by reference herein in their entireties), which, briefly, involves synthesis of overlapping oligonucleotides containing portions of the sequence encoding the fusion protein, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.4513165576vlAttorney Docket No. 2019802-0011

[0149] In some embodiments, a polynucleotide encoding an IL10 fusion protein described herein can be generated from nucleic acid sequences from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the fusion protein of interest. Such PCR amplification methods can be used to obtain nucleic acid sequences comprising the sequence encoding e.g., an IL10 polypeptide, a linker sequence, and / or an Fc region polypeptide. The amplified nucleic acid sequences can be cloned into vectors for expression in host cells and for further cloning, for example, to generate IL 10 fusion proteins.

[0150] If a clone containing a nucleic acid sequence encoding a particular IL 10 fusion protein is not available, but the sequence of an IL10 fusion protein molecule is known, a nucleic acid encoding the fusion protein can be chemically synthesized or obtained from a suitable source (e.g., a cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the proteins of interest, such as hybridoma cells selected to express a fusion protein described herein) by PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the fusion proteins. Amplified nucleic acid sequences generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0151] DNA encoding an IL10 fusion protein as described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the fusion proteins disclosed herein). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of fusion proteins in the recombinant host cells.

[0152] It is further recognized that the polynucleotide encoding an IL10 fusion protein as disclosed herein can comprise additional elements that aid in the translation of the fusion protein.4613165576vlAttorney Docket No. 2019802-0011Such sequences include, for example, Kozak sequences attached to the 5' end of the polynucleotide encoding the fusion protein. The Kozak consensus sequence is a sequence which occurs on eukaryotic mRNA that plays a role in the initiation of the translation process and has the consensus (gee)gccRccAUGG (SEQ ID NO: 14); wherein (1) a lower case letter denotes the most common base at a position where the base can nevertheless vary; (2) upper case letters indicate highly conserved bases, i.e. the 'AUGG' sequence is constant or rarely, if ever, changes, with the exception being the IUPAC ambiguity code ’R' which indicates that a purine (adenine or guanine) is normally observed at this position; and (3) the sequence in brackets ((gee)) is of uncertain significance.

[0153] In some embodiments, an TL10 fusion protein as disclosed herein comprises an IL10 leader optimized Kozak sequence as set forth in SEQ ID NO: 15 (gccaccATGGACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCAC AAACAGT) or a functional variant or fragment thereof. A functional variant or fragment of a Kozak sequence will retain the ability to increase translation of the protein when compared to the level of translation from a sequence lacking the leader. Such a functional fragment can comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 continuous nucleotides of a Kozak sequence or the sequence set forth in SEQ ID NO: 15 (see above) or in the following sequence, i.e., SEQ ID NO: 16 (gccaccATGG). In some embodiments, a functional variant can comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the Kozak sequence or the sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16.Cells and Vectors

[0154] In some embodiments provided herein are cells (e.g., host cells) expressing (e.g., recombinantly) an IL10 fusion protein as described herein and expression vectors comprising polynucleotides that encode IL10 fusion proteins as described herein. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides encoding an IL 10 fusion protein for recombinant expression in host cells.

[0155] In some embodiments, a host cell comprises nucleic acid sequences as described herein.4713165576vlAttorney Docket No. 2019802-0011

[0156] In some embodiments, a host cell is a eukaryotic cell. In some embodiments, a host cell is a mammalian cell, an insect cell, a yeast cell, a transgenic mammalian cell, or a plant cell. In some embodiments, a host cell is a prokaryotic cell. In some embodiments, a prokaryotic cell is a bacterial cell.

[0157] In some embodiments, a host cell is a mammalian cell. Such mammalian host cells include, but are not limited to, a CHO cell, a VERO cell, a BHK cell, a HeLa cell, a MDCK cell, a HEK 293 cell, a N1H 3T3 cell, a W138 cell, a BT483 cell, a Hs578T cell, a HTB2 cell, a BT2O cell, a T47D cell, a NSO cell (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), a CRL7O3O cell, a COS cell (e.g., a COS1 cell or a COS cell), a PER C6 cell, a VERO cell, a HsS78Bst cell, a HEK-293T cell, a HepG2 cell, a SP210 cell, a Rl.l cell, a B-W cell, aL-M cell, a BSC 1 cell, BSC40 cell, a YB / 20 cell, a BMTIO cell and a HsS78Bst cell.

[0158] As described herein, an "expression vector" refers to any nucleic acid construct which contains the necessary elements for the transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation, when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

[0159] A gene expression control sequence as used herein is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the coding nucleic acid to which it is operably linked. A gene expression control sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus, and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters can be used according to embodiments of the present disclosure. Promoters useful as gene expression4813165576vlAttorney Docket No. 2019802-0011 sequences of the disclosure also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, a metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

[0160] For the purposes of this disclosure, numerous expression vector systems can be employed. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Expression vectors can include expression control sequences including, but not limited to, promoters (e.g., naturally associated or heterologous promoters), enhancers, signal sequences, splice signals, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Expression vectors can also utilize DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), cytomegalovirus (CMV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites.

[0161] Commonly, expression vectors contain selection markers (e.g., ampicillin- resistance, hygromycin-resistance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences (see, e.g., Itakura et al., US Patent No. 4,704,362, the contents of which are incorporated by reference herein in their entirety, can also be used). Cells which have integrated the DNA into their chromosomes can be selected by introducing one or more markers which allow selection of transfected host cells. A marker can provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. A selectable marker gene can either be directly linked to the DNA sequences to be expressed or introduced into the same cell by cotransformation.

[0162] An example of a vector useful for optimized expression of the fusion proteins used in the methods of the present disclosure is NEOSPLA ; U.S. Patent No. 6,159,730, the contents of which are incorporated by reference herein in their entirety. This vector contains the cytomegalovirus promoter / enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase4913165576vlAttorney Docket No. 2019802-0011 exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence. Vector systems are also taught in US Patent Nos. 5,736,137 and 5,658,570, the contents of each of which is incorporated by reference herein in its entirety with respect to vector systems. This system provides high expression levels, e.g., greater than 30 pg / cell / day. Other exemplary vector systems are disclosed e.g., in US Patent No. 6,413,777, the contents of which are incorporated by reference herein in their entirety with respect to exemplary vector systems, can also be used.

[0163] In some embodiments, polypeptides of the present disclosure are expressed using polycistronic constructs. In these exemplary expression systems, multiple gene products of interest such as multiple polypeptides of multimer binding protein can be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in US Patent No. 6,193,980, the contents of which are incorporated by reference herein in their entirety with respect to IRES sequences, can also be used.

[0164] More generally, once the vector or DNA sequence encoding a polypeptide has been prepared, the expression vector can be introduced into an appropriate host cell. That is, the host cells can be transformed. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art, as discussed above. The transformed cells are grown under conditions appropriate for the production of the fusion protein and assayed for fusion protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.Pharmaceutical Compositions

[0165] An 1L10 fusion protein as disclosed herein can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise an IL 10 fusion protein and a pharmaceutically acceptable carrier.

[0166] In some embodiments, disclosed herein is a pharmaceutical composition comprising (a) an IL10 fusion protein as described herein and (b) a pharmaceutically acceptable excipient.5013165576vlAttorney Docket No. 2019802-0011

[0167] In some embodiments, disclosed herein is a pharmaceutical composition comprising (a) a composition comprising an IL10 fusion protein as described herein and (b) a pharmaceutically acceptable excipient.

[0168] In some embodiments, disclosed herein is a pharmaceutical composition comprising (a) a nucleic acid as described herein and (b) a pharmaceutically acceptable excipient.

[0169] In some embodiments, disclosed herein is a pharmaceutical composition comprising (a) a vector as described herein and (b) a pharmaceutically acceptable excipient.

[0170] In some embodiments, disclosed herein is a pharmaceutical composition comprising (a) a host cell as described herein and (b) a pharmaceutically acceptable excipient.

[0171] A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), and transmucosal. In addition, it may be desirable to administer a therapeutically effective amount of the pharmaceutical composition locally to an area in need of treatment. This can be achieved by, for example, local or regional infusion or perfusion during surgery, topical application, injection, catheter, suppository, or implant (for example, implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like. In some embodiments, the therapeutically effective amount of the pharmaceutical composition is delivered in a vesicle, such as a liposome.

[0172] In some embodiments, a therapeutically effective amount of the pharmaceutical composition can be delivered in a controlled release system. In one example, a pump can be used (see, e.g., Langer, Science 249: 1527-33, 1990; Sefton, Crit. Rev. Biomed. Eng. 14:201-40, 1987; Buchwald et al., Surgery 88:507-16, 1980; Saudek et al., N Engl. J Med. 321 :574-79, 1989; the contents of each of which is incorporated by reference herein in their entireties). In another example, polymeric materials can be used (see, e.g., Levy et al., Science 228:190-92, 1985; During et al., Ann. Neural. 25:351-56, 1989; Howard et al., J Neurosurg. 71 : 105-12, 1989; the contents of each of which is incorporated by reference herein in their entireties). Other controlled release systems, such as those discussed by Langer (Science 249: 1527-33, 1990; the contents of which are incorporated by reference herein in their entirety), can also be used.5113165576vlAttorney Docket No. 2019802-0011

[0173] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium 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 molecular weight (less than about 10 residues) polypeptides; proteins, 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0174] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose.

[0175] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as 5213165576vlAttorney Docket No. 2019802-0011 sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0176] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELS (BASF; Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0177] Sterile injectable solutions can be prepared by incorporating an IL10 fusion protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an IL10 fusion protein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.5313165576vlAttorney Docket No. 2019802-0011

[0178] For administration by inhalation, TL10 fusion proteins can be delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means.

[0179] For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, IL10 fusion proteins are formulated into ointments, salves, gels, or creams as generally known in the art. IL10 fusion proteins can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0180] In some embodiments, an IL 10 fusion protein as described herein is prepared with carriers that will protect an IL 10 fusion protein against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811, the contents of which are incorporated by reference herein in their entirety, can also be used.Uses and MethodsMethods of Making Compositions Disclosed Herein

[0181] As discussed herein, an IL 10 fusion protein of the present disclosure can be used to create new IL10 fusion proteins by modifying one or more of the IL10 polypeptide, the Fc region polypeptide, the linker, or any heterologous moiety sequence described herein. Thus, in5413165576vlAttorney Docket No. 2019802-0011 some embodiments, structural features of an TL10 fusion protein described herein may be used to create structurally related IL 10 fusion proteins that retain at least one functional property of an IL 10 fusion protein described herein, such as binding to human IL10R.

[0182] In some embodiments, disclosed herein are methods of producing an IL 10 fusion protein comprising culturing a host cell comprising a polynucleotide sequence(s) encoding the fusion protein under suitable conditions and expressing and recovering the fusion protein. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a mammalian cell, an insect cell, a fungal cell, a plant cell, a transgenic mammalian cell, or a bacterial cell. In some embodiments, the host cell includes a CHO cell, a HEK 293 cell, a NSO cell, a Per C6 cell, a BHK cell, or a COS cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the bacterial cell is Escherichia coli.

[0183] Other types of modifications include those described in the previous section. The starting material for a protein engineering method is one or more of the IL10 polypeptides or Fc region polypeptide sequences provided herein. To create an engineered (i.e., a modified) fusion protein, it is not necessary to actually prepare (i.e., express as a protein) a fusion protein having one or more of the IL 10 or Fc sequences provided herein. Rather, the information contained in the sequence(s) can be used as the starting material to create a "second generation" sequence(s) derived from the original sequence(s) and then the "second generation" sequence(s) can be prepared and expressed as an engineered fusion protein.

[0184] Accordingly, provided herein are methods for preparing a variant IL10 fusion protein comprising: (a) providing an IL10 polypeptide, an Fc region polypeptide, and a linker (e.g., a linker peptide); (b) altering at least one amino acid residue within the IL10 polypeptide, Fc region polypeptide or linker to create at least one variant IL10 fusion protein sequence; and (c) expressing the variant IL 10 fusion protein sequence as a protein.

[0185] The variant IL 10 fusion protein can exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the functional properties as disclosed herein. The functional properties of the variant IL10 fusion proteins can be assessed using standard assays available in the art.

[0186] In some embodiments, methods of engineering a variant IL 10 fusion protein are provided. For example, in some embodiments, mutations can be introduced randomly or5513165576vlAttorney Docket No. 2019802-0011 selectively along all or part of an IL10 fusion protein coding sequence and the resulting variant IL10 fusion protein can be screened for binding activity and / or other functional properties as described herein. Mutational methods have been described in the art. For example, PCT Publication WO 02 / 092780 by Short (which is incorporated by reference herein in its entirety) describes methods for creating and screening mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Further, PCT Publication WO 03 / 074679 by Lazar et al. (which is incorporated by reference herein in its entirety) describes methods of using computational screening methods to optimize physiochemical properties of proteins.

[0187] Compositions further include isolated polynucleotides that encode IL 10 fusion proteins described herein above, and variants and fragments thereof. Vectors and expression cassettes comprising the polynucleotides described herein are further disclosed. Expression cassettes will generally include a promoter operably linked to a polynucleotide and a transcriptional and translational termination region.

[0188] In constructs that include more than one processing or cleavage site, it will be understood that such sites can be the same or different.

[0189] An IL10 fusion protein that is substantially free of cellular material includes preparations of protein having less than about 30%, less than about 20%, about 10%, less than about 5%, or less than about 1% (by dry weight) of contaminating protein. When an IL 10 fusion protein of the disclosure, or a biologically active portion thereof, is recombinantly produced, optimally culture medium represents less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% (by dry weight) of chemical precursors or non- protein-of-interest chemicals.

[0190] Conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed herein. Such techniques are explained fully in the literature. See, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual" (1989);"Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan, J. E., ed. (1994)]; "Oligonucleotide Synthesis" (M.J. Gaited. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)];"Transcription And Translation" [B.D. Hames & S.I. Higgins, eds. (1984)]; "Animal Cell5613165576vlAttorney Docket No. 2019802-0011Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [TRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984), the contents of each of which are incorporated by reference herein in their entireties.

[0191] A vector which comprises the above-described polynucleotides operably linked to a promoter is also provided herein. A nucleotide sequence is "operably linked" to an expression control sequence (e.g., a promoter) when the expression control sequence controls and regulates the transcription and translation of that sequence. The term "operably linked" when referring to a nucleotide sequence includes having an appropriate start signal (e.g., ATG) in front of the nucleotide sequence to be expressed and maintaining the correct reading frame to permit expression of the sequence under the control of the expression control sequence and production of the desired product encoded by the sequence. If a gene that one desires to insert into a recombinant nucleic acid molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene. A "vector" is a replicon, such as plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. The promoter may be, or is identical to, a bacterial, yeast, insect or mammalian promoter. Further, the vector may be a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryotic viral DNA. Other numerous vector backbones known in the art as useful for expressing protein may be employed. Such vectors include, but are not limited to adenovirus, simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Moloney murine leukemia virus, DNA delivery systems, i.e. liposomes, and expression plasmid delivery systems. Further, one class of vectors comprises DNA elements derived from viruses such as bovine papilloma virus, polyoma virus, baculovirus, retroviruses or Semliki Forest virus. Such vectors may be obtained commercially or assembled from the sequences described by methods well-known in the art.

[0192] A host vector system for the production of a polypeptide which comprises the vector of a suitable host cell is provided herein. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, e.g., bacterial cells (including gram positive cells), yeast cells, fungal cells, insect cells, and animal cells. Numerous mammalian cells may be used as hosts, including, but not limited to, a mouse fibroblast cell NIH 3T3, a CHO cell, a HeLa cell, a Ltk cell, etc. Additional animal cells, such as a Rl.l cell, a B-W cell, a L-M cell, a African Green Monkey kidney cell (e.g., a COS 1 cell, a COS 7 cell, a BSC1 cell, a BSC40 cell, and a BMT105713165576vlAttorney Docket No. 2019802-0011 cell), an insect cell (e.g., a Sf9 cell), a human cell in tissue culture and a plant cell in tissue culture can also be used.

[0193] A wide variety of host / expression vector combinations may be employed in expressing the polynucleotide sequences presented herein. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage A, e.g., NM989, and other phage DNA, e.g., M13 and fdamentous single stranded phage DNA; yeast plasmids such as the 2p plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

[0194] Any of a wide variety of expression control sequences (sequences that control the expression of a nucleotide sequence operably linked to it) may be used in these vectors to express the polynucleotide sequences provided herein. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3 -phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast a-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0195] It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the polynucleotide sequences provided herein. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this disclosure. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the5813165576vlAttorney Docket No. 2019802-0011 expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

[0196] In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular nucleotide sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the nucleotide sequences to be expressed, and the ease of purification of the expression products.

[0197] In preparing an expression cassette, the various polynucleotides may be manipulated, so as to provide for the polynucleotide sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the polynucleotides, or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For example, linkers such as two glycines may be added between polypeptides. Methionine residues encoded by ATG nucleotide sequences may be added to allow initiation of gene transcription. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.

[0198] Further provided is a method of producing a polypeptide which comprises expressing a polynucleotide encoding a fusion protein disclosed herein in a host cell under suitable conditions permitting the production of the polypeptide and recovering the polypeptide so produced.Therapeutic Uses and Methods

[0199] The IL- 10 fusion proteins and methods of using same described herein have numerous in vitro and in vivo utilities involving, for example, a decrease or a suppression of an immune response. For example, IL10 fusion proteins described herein can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to decrease immunity or5913165576vlAttorney Docket No. 2019802-0011 suppress inflammation in a variety of diseases. Accordingly, provided herein are methods of treating a disease or disorder a subject in need thereof, comprising administering to the subject an IL10 fusion protein described herein such that the immune response in the subject is modified. In some embodiments, the immune response is reduced, suppressed or downregulated.

[0200] These and other methods described herein are discussed in further detail below.Inflammatory Disease or Autoimmune Disease

[0201] In some embodiments, disclosed herein are methods of treating a disease or disorder in a subject in need thereof, wherein the disease or disorder is an inflammatory disease or an autoimmune disease. In some embodiments, disclosed herein are methods of treating a disease or disorder in a subject in need thereof, wherein the disease or disorder is an inflammatory bowel disease (IBD). In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of an IL 10 fusion protein disclosed herein.

[0202] In some embodiments, an IL10 fusion protein disclosed herein is administered to a subject having an inflammatory disease (e.g., an inflammatory bowel disease) or an autoimmune disease that exhibited an inadequate response to, or progressed on, a prior treatment. In some embodiments, an IL10 fusion protein disclosed herein is administered to a subject who has not previously received (i.e., not been treated with) treatment for an inflammatory disease (e.g., an inflammatory bowel disease) or an autoimmune disease.

[0203] In some embodiments, an IL10 fusion protein disclosed herein is administered prophylactically for an inflammatory disease (e.g., an inflammatory bowel disease) or an autoimmune disease, e.g., a therapy that is intended to prevent the occurrence (or recurrence) of inflammation.

[0204] In some embodiments, an IL10 fusion protein disclosed herein is administered as a monotherapy for treatment of an inflammatory disease (e.g., an inflammatory bowel disease) or an autoimmune disease, or as the only immune-stimulating therapy for treatment of an inflammatory disease (e.g., an inflammatory bowel disease) or an autoimmune disease. In some embodiments, an IL 10 fusion protein disclosed herein is combined with a vaccination protocol for treatment of an inflammatory disease (e.g., an inflammatory bowel disease) or an6013165576vlAttorney Docket No. 2019802-0011 autoimmune disease. In some embodiments, an IL10 fusion protein disclosed herein is combined with an antibody used for treatment of an inflammatory disease (e.g., an inflammatory bowel disease) or an autoimmune disease.

[0205] In some embodiments, the inflammatory disease or an autoimmune disease includes type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematous, lupus nephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft versus host disease, psoriasis, alopecia areata, HCV- induced vasculitis, Sjogren’s syndrome, Pemphigus, Ankylosing Spondylitis, Behcet's Disease, Wegener's Granulomatosis, Takayasu's Disease, Autoimmune Hepatitis, Sclerosing Cholangitis, Gougerot-sjbgren, chronic obstructive pulmonary disease (COPD), or Macrophage Activation Syndrome.Co-Administration with a Second Agent

[0206] As previously described, an IL 10 fusion protein disclosed herein can be coadministered with one or other more therapeutic agents, e.g., an immunosuppressive agent. An IL10 fusion protein disclosed herein can be linked to the agent (as an immuno-complex) or can be administered separately from the agent. In the latter case (separate administration), an IL 10 fusion protein disclosed herein can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g., anti-inflammation therapy.

[0207] Provided herein are methods of combination therapy in which an IL 10 fusion protein disclosed herein is co-administered with one or more additional agents (a second therapeutic agent), e.g., small molecule drugs, antibodies or antigen binding portions thereof, that are effective in suppressing immune responses in a subject. Such therapeutic agents include, among others, drugs containing mesalamine (including sulfasalazine and other agents containing 5-aminosalicylic acid (5-ASA), such as olsalazine and balsalazide), non-steroidal antiinflammatory drugs (NSAIDs), analgesics, corticosteroids (e.g., predinisone, hydrocortisone), immunosuppressants (such as 6-mercaptopurine, methotrexate, azathioprine and cyclosporine A), Orencia®, TNF -inhibitors (including adilimumab (Humira®), etanercept (Enbrel®) and infliximab (Remicade®)).6113165576vlAttorney Docket No. 2019802-0011

[0208] In some embodiments, a combination of a fusion protein of the present disclosure and a second agent discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with the fusion protein of the present disclosure and the second agent in a pharmaceutically acceptable carrier. In some embodiments, the combination of an IL10 fusion protein of the present disclosure and the second agent can be administered sequentially. The two agents can be administered to a subject at a time that optimally promotes the therapeutic effect(s), e.g., the second agent can be administered about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 3 days, about 5 days, about 7 days, or about one or more weeks after administration of the first agent.Kits

[0209] Provided herein, among other things, are kits comprising an IL10 fusion protein described herein, and instructions for use and / or administration. In some embodiments, a kit comprises an IL10 fusion protein described herein, a pharmaceutically acceptable carrier, and instructions for use and / or administration. In some embodiments, a kit comprises a nucleic acid sequence encoding an IL 10 fusion protein as described herein. In some embodiments, a kit comprises a nucleic acid sequence encoding an IL 10 fusion protein described herein and instructions for use and / or administration. In some embodiments, a kit comprises a nucleic acid sequence encoding an IL 10 fusion protein described herein, a pharmaceutically acceptable carrier, and instructions for use and / or administration.

[0210] Also provided are kits for use in the various methods disclosed herein. In some embodiments, a kit comprises instructions for use in any method described herein. Instructions can comprise a description of administration of a first and second pharmaceutical composition to a subject to achieve an intended activity in a subject. A kit may further comprise a description of selecting a human suitable for treatment based on identifying whether the human is in need of the treatment. In some embodiments, the instructions comprise a description of administering an IL10 fusion protein described herein to a subject.6213165576vlAttorney Docket No. 2019802-0011

[0211] Instructions relating to administering a dose comprising an IL10 fusion protein described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions supplied in kits as described herein are typically written instructions on a label or package insert. A label or package insert indicates that pharmaceutical compositions are used for treating or preventing a disease, disorder, or condition described herein (e.g., an autoimmune disease, disorder, or condition in a subject.

[0212] Kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierce able by a hypodermic injection needle). A container may also have a sterile access port.

[0213] Kits optionally may provide additional components such as buffers and interpretive information. Normally, a kit can include a container, and a label or package insert(s) on or associated with the container. In some embodiments provided herein are articles of manufacture comprising contents of kits described herein.

[0214] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.EXAMPLESExample 1. Construction of IL10 fusion proteins

[0215] An IL10 fusion protein that forms a stable, monomeric IL-10, was constructed by fusing (from N-terminus to C-terminus) a human IgGl ,3f Fc region polypeptide (SEQ ID NO: 3), a Gly-Ser rich polypeptide linker (GGGSGGGGSGGGGS, SEQ ID NO: 4), and a modified human IL10 polypeptide (SEQ ID NO: 2) (referred to herein as "Fusion protein 1", see SEQ ID6313165576vlAttorney Docket No. 2019802-0011NO: 1). An illustration of certain structural features of Fusion protein 1 are depicted in FTGs. 1- 2.

[0216] A further IL 10 fusion protein that forms a stable, dimeric IL- 10, was constructed by fusing (from N-terminus to C-terminus) a human IgG1.3f Fc domain (SEQ ID NO: 3), a 22- amino acid Gly-Ser rich polypeptide linker sequence, and wild-type human IL- 10 (referred to herein as "Fusion Protein 2; see SEQ ID NO: 8).

[0217] The IgG1.3f Fc domain was chosen to provide reduced antibody -dependent cellular cytotoxicity (ADCC) function while a linker serves to separate the IL10 monomer from the Fc domain. The IL10 monomeric form of Fusion protein 1 was generated by inserting a 6- amino acid spacer (GGGSGG, SEQ ID NO: 5) into the polypeptide sequence of wildtype IL 10 between helices D and E (FIG. 2). Such insertion changes the quintenary assembly of the IL 10 domain from a “domain swapped dimer” to instead form a soluble monomeric “modified monomer” version of IL10 (FIG. 1).

[0218] The fusion proteins described herein can be prepared and expressed using routine procedures known in the art.Example 2. In vitro analysis of the myeloid selectivity of Fusion protein 1

[0219] For in vitro myeloid selectivity studies, STAT3 phosphorylation induced by incubation in the presence of either Fusion protein 1 or Fusion protein 2 (as a reference) was measured in human Monocytes (CD14+), T cells (CD3+), and B cells (CD20+) using flow cytometry. A flow cytometry panel was prepared as follows: CD20 [L20; Thermo Fisher Scientific], pSTAT3 (pY705) [4 / P-STAT3; BD Biosciences], CD3 [SP34-2; BD Biosciences], CD66abce [TET2; Miltenyi Biotec], and CD14 [M5E2; BD Biosciences]. Briefly, human whole blood from healthy donors was stimulated with either Fusion protein 1 or Fusion protein 2 or controls for 15 minutes. Stimulation was stopped, and RBCs lysed by adding pre-warmed Phosflow Lyse / Fix buffer [BD Biosciences]. Following fixation and RBC lysis, samples were washed with FACS buffer (Dulbecco’s phosphate-buffered saline (DPBS)) [Gibco] supplemented with 2% Fetal Bovine Serum (FBS) [Gibco], Cells were then permeabilized using Phosflow Buffer III [BD Biosciences] for 1 hour and washed with FACS buffer. Washed and6413165576vlAttorney Docket No. 2019802-0011 permeabilized cells were then FC blocked for 10 minutes at room temp [422302; Biolegend]. Following blocking, cells were stained with all listed antibodies simultaneously. After a 1 hour incubation at room temperature cells were washed and resuspended in FACS buffer, acquired on a FACSCantoXTM flow cytometer, and analyzed using FlowJo. Fusion protein 2 resulted in pSTAT3 induction with similar Ymax to recombinant IL-10 (FIG. 3A), while Fusion protein 1 showed myeloid selectivity and maintained 74% of the Ymax achieved with recombinant IL-10 in monocytes but only 16% in T cells (FIG. 3B). These data show that Fusion protein 1 is myeloid selective.Example 3. In vitro analysis of the ability of Fusion protein 1 to induce IFNy secretion on primary CD4 and CD8+ T cells

[0220] The induction of IFNy by recombinant human IL-10 (control; hIL-10; R&D Systems), Fusion protein 1, and Fusion protein 2 on primary human CD4+ or CD8+ T cells was measured using Perkin Elmer IFNg AlphaLISA detection kit. Briefly, PBMCs were isolated from human whole blood from healthy donors using density gradient centrifugation with Lymphoalyte H (Cedarlane). CD4+ or CD8+ T cells were negatively isolated using a CD4+ or a CD8+ T cell Isolation kit (Stem Cell). Isolated CD4+ or CD8+ T cells were stimulated with CD3 / CD28 T cell activator complex (Stem Cell) in complete media (Stem Cell) with 20 U / mL of recombinant human IL-2 (R&D Systems) for 72 hours at 37°C in 5% CO2. After incubation, and prior to stimulation with Fusion protein 1, Fusion protein 2, or the control, cells were washed with Phosphate-buffered saline (Gibco), plated in complete media with or without 20 U / mL recombinant human IL-2 (R&D Systems) for an additional 72 hours. Supernatants were analyzed for IFNy using Perkin Elmer (now Revvity) IFNy AlphaLISA detection kit. The data demonstrated that recombinant human IL- 10 (FIG. 4A) and Fusion protein 2 (FIG. 4B) induced fFNy secretion by primary human CD4+ (data not shown) or CD8+ T cells in a dose dependent manner. Notably, the data demonstrated that Fusion protein 1 (FIG. 4C) did not induce fFNy secretion by primary human CD4+ (data not shown) or CD8+ T cells. These data therefore demonstrated that Fusion protein 1 does not induce IFNy in CD4+ (data not shown) or CD8+ T cells (FIG. 4C).6513165576vlAttorney Docket No. 2019802-0011Example 4. In vitro analysis of the ability of Fusion protein 1 to suppress LPS-induced TNF

[0221] A human whole blood lipopolysaccharide (LPS)-induced cytokine suppression assay was performed to measure the ability of Fusion protein 1 to suppress LPS-induced TNF. Heparinized healthy human whole blood was diluted (1 : 1.6 with Dulbecco's Modified Eagle Medium (DMEM)) [Gibco] and added to the wells of a 96-well u bottom plate. Experimental wells were then stimulated with 100 ng / mL of lipopolysaccharide (LPS) [L6143; Sigma] and treated with increasing concentrations of Fusion protein 1 or Fusion protein 2 (FIG. 5). After mixing, all samples were incubated (37°C; 5% CO2) for 24 hours. Immediately after incubation, the samples were centrifuged at 500 x g for 10 minutes and the resulting supernatants were transferred to a clean 96-well u bottom plate prior to storage at -80°C. Within 7 days, supernatants were thawed, and TNF concentrations quantified using Perkin Elmer (now Revvity) TNF AlphaLISA detection kit. The data demonstrated that Fusion protein 1 fully inhibits LPS- induced TNF production in human whole blood with an IC50 comparable to Fusion protein 2.Example 5. Assessment of the efficacy of Fusion protein 1 in a T cell transfer colitis model

[0222] Fusion protein 1 and Fusion protein 2 were tested in a T cell transfer colitis model. Briefly, 5 x e5 naive CD4+ T cells were transferred intraperitoneally intoSCID Balb / c mice (10 mice / group). Starting at day 1 post transfer, Fusion protein 1 was injected at 0.03, 0.1 and 1 mpk at weekly intervals. A similar dosing regimen was followed for Fusion protein 2 at 1 mpk. The study was terminated at the end of week 5. In addition to body weight measurements, the colons were collected for assessment by histology (FIG. 6), spleens were collected for assessment of IFNy production by resident T cells (data not shown), and blood was collected for pharmacokinetics (data not shown), hematological analysis (data not shown), and assessment of cytokine levels (data not shown). The data demonstrated that Fusion protein 1 demonstrated superior efficacy with over 75% reduction in histology scores in the distal colon at the end of the study in compassion to only 21% reduction in histology scores when compared to Fusion protein 2 at 13 -fold higher exposure (FIG. 6), indicating the robust efficacy of Fusion protein 1 in chronic T cell transfer colitis.6613165576vlAttorney Docket No. 2019802-0011Example 6. Assessment of the effect of Fusion protein 1 on TFNy levels in splenic CD4+ T cells in a T cell Transfer colitis model

[0223] The effect of Fusion protein 1 and Fusion protein 2 on IFNy levels in splenic CD4+ T cell were analyzed in a T cell transfer colitis model. Briefly, 5xe5 naive CD4+ T cells were transferred intraperitoneally into SCID Balb / c mice (10 mice / group). Starting at day 1 post transfer, Fusion protein 1 was injected at 0.03, 0.1 and 1 mpk at weekly intervals. A similar dosing regimen was followed for Fusion protein 2 at 1 mpk. The study was terminated at the end of week 5 and spleens were collected, single cell suspensions prepared and stimulated with Cell Stimulation Cocktail (eBioscience) over night. Subsequently, a Protein Transport Inhibitor Cocktail (eBioscience) was added for 3 hours and IFNy induction was measured in mouse CD4+ T cells using flow cytometry. The flow cytometry panel was as follows: NK1.1 [PK136; Biolegend], Ly-6G [1A8; Biolegend], CD4 [RM4-5; Invitrogen], CDl lb [MI / 70; BD Biosciences], CD3 [17A2; BD Biosciences], CD45R [RA3-6B2; BD Biosciences], and IFNg [XMG1.2; Invitrogen], Following RBC lysis and surface staining, the samples were washed with FACS buffer (Dulbecco’s phosphate-buffered saline (DPBS)) [Gibco] supplemented with 2% Fetal Bovine Serum (FBS) [Gibco], Cells were then fixed and permeabilized using BD Cytofix / CytoPerm kit [BD Biosciences] for 15 minutes at room temperature. Cells were then stained with the intracellular antibodies for 1 hour at room temperature. Then the cells were washed and resuspended in FACS buffer, acquired on a FACSCantoXTM flow cytometer, and analyzed using FlowJo. Fusion protein 2 treatment resulted in a significant induction of IFNy in splenic CD4+ T cells (FIG. 7). Notably, treatment with Fusion protein 1 resulted in no such induction (FIG. 7). Importantly, these data differentiated the effect of Fusion protein 1 from the effect of Fusion protein 2, demonstrating that Fusion protein 1 does not induce IFNy CD4+Thl cells in the spleen.6713165576vlAttorney Docket No. 2019802-0011Example 7. Assessment of the effect of Fusion protein 1 on platelet levels in a T cell transfer colitis model

[0224] The effect of Fusion protein 1 and Fusion protein 2 on platelet numbers was analyzed in a T cell transfer colitis model. Briefly, 5xe5 naive CD4+ T cells were transferred intraperitoneally into SCID Balb / c mice (10 mice / group). Starting at day 1 post transfer, Fusion protein 1 was injected at 0.03, 0.1 and 1 mpk at weekly intervals. A similar dosing regimen was followed for Fusion protein 2 at 1 mpk. The study was terminated at the end of week 5 and EDTA treated blood was collected for CBC analysis and platelet counts on an Advia 2120i (Siemens). Treatment with Fusion protein 2 resulted in a significant drop in platelet counts (FIG. 8). Notably, the data for Fusion protein 1 demonstrated no negative effect on platelet numbers (FIG. 8). Importantly, these data further differentiated the effect of Fusion protein 1 from the effect of Fusion protein 2, demonstrating that Fusion protein 1 does not reduce platelet levels.6813165576vl

Claims

Attorney Docket No. 2019802-0011What is claimed is:

1. An IL10 fusion protein comprising an amino acid sequence at least about 99% identical to SEQ ID NO: 1.

2. An IL10 fusion protein consisting of (a) a first polypeptide consisting of an amino acid sequence at least about 99% identical to SEQ ID NO: 1 and (b) a second polypeptide consisting of an amino acid sequence at least about 99% identical to SEQ ID NO: 1, wherein the first polypeptide and the second polypeptide each comprise an Fc region, and wherein the first polypeptide and the second polypeptide are associated with each other by at least one covalent disulfide bond and / or at least one non-covalent interaction between the Fc region of the first polypeptide and the Fc region of the second polypeptide.

3. An IL10 fusion protein comprising the amino acid sequence as set forth in SEQ ID NO: 1.

4. An IL10 fusion protein consisting of (a) a first polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 1 and (b) a second polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 1, wherein the first polypeptide and the second polypeptide each comprise an Fc region, and wherein the first polypeptide and the second polypeptide are associated with each other by at least one covalent disulfide bond and / or at least one non- covalent interaction between the Fc region of the first polypeptide and the Fc region of the second polypeptide.

5. An IL 10 fusion protein comprising:(a) an IL10 polypeptide comprising an amino acid sequence at least about 95% identical to SEQ ID NO: 2;(b) an Fc polypeptide comprising an amino acid sequence at least about 95% identical to SEQ ID NO: 3; and(c) a linker operably linked to the IL 10 polypeptide and to the Fc polypeptide,6913165576vlAttorney Docket No. 2019802-0011 wherein the C-terminus of the Fc polypeptide is linked to the N-terminus of the linker and wherein the N-terminus of the IL10 polypeptide is linked to the C-terminus of the linker.

6. The IL10 fusion protein of claim 5, wherein the IL10 polypeptide comprises an amino acid sequence at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100%> identical to SEQ ID NO: 2.

7. The IL 10 fusion protein of claim 5 or 6, wherein the IL 10 polypeptide comprises the amino acid sequence according to SEQ ID NO: 2.

8. The IL10 fusion protein of any one of claims 5-7, wherein the IL10 polypeptide consists of the amino acid sequence according to SEQ ID NO: 2.

9. The IL10 fusion protein of claim 5 or 6, wherein the Fc polypeptide comprises an amino acid sequence at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 3.

10. The IL10 fusion protein of any one of claims 5-9, wherein the Fc polypeptide comprises the amino acid sequence according to SEQ ID NO: 3.

11. The IL10 fusion protein of any one of claims 5-10, wherein the Fc polypeptide consists of the amino acid sequence according to SEQ ID NO: 3.

12. The IL10 fusion protein of any one of claims 5-9, wherein the linker consists of 14 amino acids, 15 amino acids, or 16 amino acids.

13. The IL10 fusion protein of any one of claims 5-12, wherein the linker comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 4.7013165576vlAttorney Docket No. 2019802-001114. The TL10 fusion protein of any one of claims 1 to 13, wherein the fusion protein further comprises a heterologous moiety.

15. The IL10 fusion protein of any one of claims 1 to 14, wherein the fusion protein is more myeloid selective than an IL10 polypeptide consisting of SEQ ID NO: 6 or an IL10 fusion polypeptide consisting of SEQ ID NO: 8.

16. The IL10 fusion protein of any one of claims 1, 3, or 5 to 15, wherein the fusion protein is a monomer.

17. The IL10 fusion protein of any one of claims 1, 3, or 5 to 15, wherein the fusion protein is a dimer comprising two monomers, and wherein the two monomers are associated with each other via their Fc regions.

18. The IL10 fusion protein of claim 17, wherein the Fc regions of the two monomers are associated with each other by covalent disulfide bonds and / or non-covalent interactions.

19. The IL10 fusion protein of claim 17, wherein the two monomers are identical.

20. The IL 10 fusion protein of claim 17, wherein the two monomers are different.

21. The IL10 fusion protein of claim 19, wherein each of the two monomers comprises the amino acid sequence of SEQ ID NO: 1 or 2.

22. The IL 10 fusion protein of claim 20, wherein one of the two monomers comprises the amino acid sequence of SEQ ID NO: 1 or 2.

23. A composition comprising the IL 10 fusion protein of any one of claims 1-22.

24. A nucleic acid that encodes the IL10 fusion protein of any one of claims 1-22.7113165576vlAttorney Docket No. 2019802-001125. A vector comprising the nucleic acid of claim 24.

26. A host cell comprising the nucleic acid of claim 24.

27. The host cell of claim 26, wherein the host cell is a mammalian cell.

28. A pharmaceutical composition comprising (a) the 1L10 fusion protein of any one of claims 1-22, the composition of claim 23, the nucleic acid of claim 24, the vector of claim 25, or the host cell of claim 26 or 27; and (b) a pharmaceutically acceptable excipient.

29. A kit comprising the fusion protein of any one of claims 1-22 and instructions for administering the fusion protein to a subject in need thereof.

30. A method of producing the IL10 fusion protein of any one of claims 1-22, comprising: culturing the host cell of claim 26 or 27 under suitable conditions and recovering the fusion protein.

31. The method of claim 30, wherein the host cell is a mammalian cell.

32. A method of treating a disease or a disorder for a subject in need thereof, comprising administering to the subject an effective amount of the fusion protein of any one of claims 1-22, the composition of claim 23, or the pharmaceutical composition of claim 28.

33. The method of claim 32, wherein the disease or the disorder is an inflammatory disease or an autoimmune disease.

34. The method of claim 33, wherein the inflammatory disease or the autoimmune disease is type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematous, lupus nephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft versus host disease, psoriasis, alopecia areata, HCV- induced vasculitis, Sjogren’s syndrome, Pemphigus, Ankylosing Spondylitis, Behcet's Disease,7213165576vlAttorney Docket No. 2019802-0011Wegener's Granulomatosis, Takayasu's Disease, Autoimmune Hepatitis, Sclerosing Cholangitis, Gougerot- sjogren, chronic obstructive pulmonary disease (COPD) and Macrophage Activation Syndrome, inflammatory bowel disease, or any combination thereof.

35. The method of claim 33 or 34, wherein the inflammatory disease or the autoimmune disease is an inflammatory bowel disease.

36. The method of claim 33 or 34, wherein the inflammatory disease or the autoimmune disease is Crohn's disease.

37. The method of claim 33 or 34, wherein the inflammatory disease or the autoimmune disease is ulcerative colitis.

38. The method of claim 33 or 34, wherein the inflammatory disease or the autoimmune disease is COPD.

39. A method of treating a disease or a disorder for a subject in need thereof, by administering an IL10 fusion protein comprising:(a) a means for specifically binding a human IL-10 receptor; and(b) an IL10 polypeptide comprising a spacer, an Fc polypeptide and a linker operably linked to the IL 10 polypeptide and to the Fc polypeptide, wherein said IL 10 fusion protein has increased myeloid selectivity as compared to an IL 10 fusion protein without said spacer, as determined using an in vitro STAT3 phosphorylation assay.

40. A method of treating a disease or a disorder for a subject in need thereof, by administering an IL10 fusion protein comprising:(a) a means for specifically binding a human IL- 10 receptor; and(b) an IL10 polypeptide comprising a spacer, an Fc polypeptide and a linker operably linked to the IL 10 polypeptide and to the Fc polypeptide, wherein said IL 10 fusion protein has decreased IFNy induction as compared to an IL 10 fusion protein without said spacer, as determined using an in vitro IFNy induction assay.7313165576vlAttorney Docket No. 2019802-001141. The method of claim 39 or 40, wherein the C-terminus of the Fc polypeptide is linked to the N-terminus of the linker and wherein the N-terminus of the IL10 polypeptide is linked to the C-terminus of the linker.

42. The method of any one of claims 39 to 41, wherein the disease or the disorder is an inflammatory disease or an autoimmune disease.

43. The method of claim 42, wherein the inflammatory disease or the autoimmune disease is type 1 diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematous, lupus nephritis, cutaneous lupus, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis or systemic sclerosis, graft versus host disease, psoriasis, alopecia areata, HCV- induced vasculitis, Sjogren’s syndrome, Pemphigus, Ankylosing Spondylitis, Behcet's Disease, Wegener's Granulomatosis, Takayasu's Disease, Autoimmune Hepatitis, Sclerosing Cholangitis, Gougerot- sjbgren, chronic obstructive pulmonary disease (COPD) and Macrophage Activation Syndrome, inflammatory bowel disease, or any combination thereof.

44. The method of claim 42 or 43, wherein the inflammatory disease or the autoimmune disease is an inflammatory bowel disease.

45. The method of claim 42 or 43, wherein the inflammatory disease or the autoimmune disease is Crohn's disease.

46. The method of claim 42 or 43, wherein the inflammatory disease or the autoimmune disease is ulcerative colitis.

47. The method of claim 42, wherein the inflammatory disease or the autoimmune disease is COPD.

48. Use of the fusion protein of any one of claims 1-22, the composition of claim 23, or the pharmaceutical composition of claim 28 for the treatment of a disease or a disorder.7413165576vlAttorney Docket No. 2019802-001149. Use of the fusion protein of any one of claims 1-22, the composition of claim 23, or the pharmaceutical composition of claim 28 in the manufacture of a medicament for the treatment of a disease or a disorder.

50. The fusion protein of any one of claims 1-22, the composition of claim 23, or the pharmaceutical composition of claim 28 for use as a medicament.

51. The fusion protein of any one of claims 1-22, the composition of claim 23, or the pharmaceutical composition of claim 28 for use in the treatment of a disease or a disorder.7513165576vl

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