Il-10 variant molecules and methods of treating inflammatory diseases and tumors

Abstract

Provided are compositions or formulations comprising variant IL-10 molecules, fusion proteins, and chimeric proteins thereof that are useful for the treatment of cancer, inflammatory diseases or disorders, and autoimmune diseases or disorders.SOLUTION: Variant forms of interleukin 10 (IL-10) are provided that contain modifications to the IL-10 receptor binding region and / or domains involved in the interdomain angles present in the IL-10 molecule. It has been discovered that by altering one or both of these domains in IL-10, the resulting biological function of the IL-10 receptor can be tailored or modulated to elicit a particular biological response.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 814,669, filed March 6, 2019, U.S. Provisional Patent Application No. 62 / 899,504, filed September 12, 2019, and U.S. Provisional Patent Application No. 62 / 962,332, filed January 17, 2020, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Introduction The present application relates to variant forms of interleukin-10 (IL-10) containing modifications to the IL-10 receptor binding region and / or the domains involved in the interdomain angle present in the IL-10 molecule. By modifying one or both of these domains in IL-10, the inventors surprisingly discovered that the biological function of the resulting IL-10 receptor can be tailored or modulated to elicit specific biological responses. The present application also relates to IL-10 or IL-10 variant molecules with extended half-life, including non-protein-based serum extension moieties as well as protein-based extension modalities. The present application also relates to fusion proteins comprising IL-10 variant molecules. [Background technology]

[0003] background IL-10 mediates (i) pro-inflammatory cytokine secretion by monocytes / macrophages in response to lipopolysaccharide and (ii) CD4 + It has been described as a cytokine synthesis inhibitor due to its ability to inhibit both interleukin 2 (IL-2) secretion and proliferation of T cells. When viral analogs of IL-10 were discovered and reported to share similar or identical functions with human IL-10, it was speculated that these viral analogs of IL-10 enhanced viral virulence by adopting the functions of the inhibitory cytokine found in the human genome.

[0004] Further investigation of the inhibitory effects of IL-10 was made possible by the generation of IL-10 knockout mice that develop chronic enterocolitis. Data obtained from these mice showed that IL-10 knockout mice have a reduced number of cells, primarily monocytes / macrophages and CD4 + We clearly demonstrate that T cells secrete chronic inflammatory cytokines, leading to the development of severe inflammation throughout the gastrointestinal tract, consistent with earlier in vitro observations.

[0005] Taken together, the data indicated that IL-10 played a major role in suppressing inflammation. In particular, patients lacking functional IL-10 receptors or the ability to produce IL-10 showed an increased predisposition to developing inflammation-associated gastrointestinal diseases.

[0006] Multiple clinical trials have evaluated the anti-inflammatory function of IL-10 in the context of psoriasis, rheumatoid arthritis, and Crohn's disease. Generally, recombinant human IL-10 (rHuIL-10) treatment has been found to be safe but lacking in efficacy. Notably, treatment of Crohn's disease patients with rHuIL-10 results in an inverse dose-response response, with low doses moderately inhibiting inflammation and higher doses losing the inhibitory effect. A rigorous analysis of the final Crohn's disease study found that patients receiving 10 and 20 μg / kg of rHuIL-10 exhibited increased serum concentrations of interferon gamma (IFNγ) and neopterin. IFNγ is known to exacerbate the pathogenesis of inflammatory bowel disease and Crohn's disease. Data suggest that at high doses, treatment with IL-10 induces IFNγ, which exacerbates inflammatory disease.

[0007] Further analysis of the effects of IL-10 in healthy humans suggests that administering IL-10 before exposure to the pro-inflammatory factor lipopolysaccharide (LPS) inhibits the production of pro-inflammatory cytokines. However, administering rHuIL-10 after exposure to LPS enhanced the secretion of pro-inflammatory cytokines. Because LPS is a product of both normal and foreign intestinal bacteria in patients with inflammatory bowel disease (IBD), these patients never "clear" from LPS and thus never reach a state where IL-10 treatment can be applied before LPS. Therefore, these data suggest that patients with Crohn's disease and other inflammatory diseases will never derive therapeutic benefit from IL-10 treatment.

[0008] Accumulating data showing that IL-10 activates the immune system to induce antitumor responses has added further confusion to the role of IL-10. Early data suggested that IL-10 activated NK cells, but further investigations have shown that IL-10 treatment activates CD8 + IL-10 treatment was found to inhibit tumor growth in a T cell- and IFNγ-dependent manner. Further investigations have shown that IL-10 treatment inhibits FoxP3 + CD4 + It has been found to inhibit the proliferation of regulatory T cells and enhance the elimination of Kupffer cells, all functions that suggest that IL-10 is a potent immune stimulator rather than a suppressor. Finally, these stimulatory activities, where confirmed, have been in clinical studies of tumor patients treated with PEGylated IL-10.

[0009] Taken together, these data demonstrate that IL-10 treatment of patients with autoimmune-related inflammation fails to induce therapeutic benefit, suggesting that IL-10 is not a pan-immunosuppressant. Consistent with this, treatment of cancer patients with (PEG)IL-10, similar to IL-10-treated Crohn's disease patients, resulted in potent and therapeutically useful immune activation, particularly a dose-dependent induction of serum IFNγ, indicating that IL-10 is a potent immune stimulator.

[0010] Why viruses acquire IL-10 sequences remains unclear. Further analysis of both Epstein-Barr virus (EBV-IL10) and cytomegalovirus (CMV-IL10) homologs suggests that these viruses alter the native IL-10 sequence in two major ways. EBV-IL10 maintains a similar three-dimensional angle of homodimerization, resulting in a specific angle of ligand-receptor interaction, but its affinity for the IL-10 receptor appears to be substantially reduced. CMV-IL10 exhibits increased affinity for the IL-10 receptor, but the angle of interaction with the IL-10 receptor is substantially altered. While the EBV-IL10 and CMV-IL10 sequences maintain approximately 80% and 27% homology to native IL-10, respectively, each viral homolog possesses a completely different IL-10 receptor affinity and receptor binding angle, and each viral homolog appears to exert anti-inflammatory functions highly similar to native IL-10. Therefore, it is unclear how the affinity for the IL-10 receptor and / or the angle of receptor interaction influence the subsequent downstream signaling of IL-10. Summary of the Invention [Means for solving the problem]

[0011] Summary of Various Preferred Embodiments The present application generally relates to novel IL-10 variant molecules that modulate IL-10 receptor signaling. Accordingly, the present application relates to IL-10 variant molecules incorporating modifications to the structure of the IL-10 molecule, resulting in novel IL-10 variant molecules with altered IL-10 receptor binding affinity and / or altered IL-10 interdomain angles. The inventors surprisingly discovered that modifying IL-10 at key domains that affect IL-10 receptor affinity and / or IL-10 interdomain angles creates IL-10 receptor agonists that can be used in the treatment of immune diseases, inflammatory diseases or conditions, and cancer. Furthermore, IL-10 variant molecules can also have increased serum half-lives by incorporating the variant IL-10 molecule as part of a fusion protein, e.g., various antibody domains (Fc or variable domains), without preventing IL-10 or IL-10 variant monomers from forming IL-10 homodimers.

[0012] In certain embodiments, the IL-10 variant molecule is a modified human IL-10. In other embodiments, the IL-10 variant molecule is a modified murine IL-10. In preferred embodiments, the IL-10 variant molecule is a modified viral homolog of IL-10. In a more preferred embodiment, the viral IL-10 homolog is CMV-IL10. In a most preferred embodiment, the viral IL-10 homolog is EBV-IL10.

[0013] In further embodiments, the IL-10 variant molecule incorporates at least one or more amino acid additions, deletions, or substitutions within the receptor-binding region. In yet other embodiments, the IL-10 variant molecule incorporates at least one or more amino acid additions, deletions, or substitutions in the region involved in forming the interdomain angle of IL-10. In preferred embodiments, the IL-10 variant molecule incorporates one or both of the modifications to the receptor-binding domain and / or the region involved in the interdomain angle. In the most preferred embodiment, the IL-10 variant molecule incorporates one or both modifications in the EBV-IL10 protein molecule.

[0014] In various embodiments, the IL-10 variant molecule has enhanced affinity for the IL-10 receptor or receptor complex compared to wild-type IL-10. In other embodiments, the IL-10 variant molecule has reduced affinity for the IL-10 receptor or receptor complex compared to wild-type IL-10. In other embodiments, the IL-10 variant molecule forms a narrow or restricted interdomain angle compared to wild-type IL-10, more preferably EBV-IL10. In another embodiment, the IL-10 variant molecule forms a wide or relaxed interdomain angle compared to wild-type IL-10, more preferably EBV-IL-10.

[0015] In still further embodiments, the EBV IL-10 variant molecule incorporates at least one or more amino acid additions, deletions, or substitutions within the receptor binding region located in helix A, the AB loop, and / or helix F ("site 1") of EBV IL-10. Modifications to the receptor binding domain region may, in certain embodiments, increase or enhance affinity for the IL-10 receptor or receptor complex. In certain other embodiments, modifications to the receptor binding region may attenuate or decrease affinity for the IL-10 receptor or receptor complex.

[0016] In further embodiments, the EBV IL-10 variant molecule incorporates at least one or more amino acid additions, deletions, or substitutions within the EBV IL-10 region involved in interdomain angle formation. In a preferred embodiment, the modification may occur in the DE loop of EBV IL-10. Modifications in the DE loop result in EBV IL-10 variant molecules with restricted or relaxed interdomain angles.

[0017] In other aspects, the IL-10 variant molecule is a chimeric or fusion molecule. In one embodiment, a chimeric or fusion protein will contain one or more domains from different proteins or mutations that confer characteristics of another protein within a single protein. In a preferred embodiment, the chimeric or fusion protein will contain an IL-10 variant molecule, such as albumin, an enzyme, a glycosyltransferase, a galactosyltransferase, an IgG hinge region (e.g., an Fc region), one or more variable domains of one or more antibodies (e.g., but not limited to, a variable heavy chain or a variable light chain), a cytokine (e.g., IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, GM-CSF, or the like). The fusion protein may comprise a first portion comprising an IL-10 variant molecule described herein fused to another molecule, including, but not limited to, interferon-α, -β, -γ, TGF-β, and tumor necrosis factor-α, -β), labels, drugs, chemotherapeutic agents, radioisotopes, and half-life extenders (e.g., hydroxyethyl starch (HES), polysialic acid, heparosan polymer, elastin-like polypeptide, and hyaluronic acid). In yet another embodiment, the IL-10 variant molecule is fused to one or more antibody heavy or light chain variable regions. Fusion proteins may contain one or more linkers covalently linking different portions of the fusion protein. Fusion proteins may also form non-covalently bound complexes with other fusion proteins of the same type. Such a fusion protein would allow monomers of IL-10, or monomers of variant IL-10 molecules, to associate together into functional homodimers of IL-10 or variant IL-10.

[0018] In other embodiments, the variant IL-10 molecule is part of an engineered fusion protein. The fusion protein comprises at least one monomer of IL-10 or an IL-10 variant molecule conjugated to a linker or spacer at one end of the fusion protein, where one or more spacers are used to link the various portions of the fusion protein, which is then conjugated to at least one other molecule conjugated to the other end, where the molecule is selected from at least one cytokine or a monomer thereof, a therapeutic agent, a label, a serum half-life extension molecule, or a protein (e.g., but not limited to, various portions of a receptor, ligand, or antibody). In a preferred embodiment, the fusion protein comprises a monomer of IL-10 or a monomer of an IL-10 variant molecule conjugated to at least one heavy and / or light chain variable region via a linker or spacer. In a most preferred embodiment, the fusion protein comprises a monomer of EBV IL-10 or a monomer of an EBV IL-10 variant molecule conjugated to at least one heavy and / or light chain region via a linker or spacer. In a most preferred embodiment, a monomer of EBV IL-10 or an EBV IL-10 variant molecule is conjugated to one heavy chain variable region and one light chain variable region, and the monomers together form a dimeric complex. The monomer of EBV IL-10 or an EBV IL-10 variant molecule can be conjugated to either the amino or carboxy terminus of the heavy or light chain variable region.

[0019] In certain embodiments, a therapeutic amount of the IL-10 variant molecule, fusion protein, or chimeric protein thereof of the present application is administered to a subject suffering from an inflammatory disease or condition, such as, but not limited to, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, nonalcoholic steatohepatitis (NASH), and nonalcoholic fatty liver disease (NAFLD). In another embodiment, a therapeutic amount of the IL-10 variant molecule, fusion protein, or chimeric protein thereof of the present application is administered to a subject suffering from cancer. Treatment of subjects with more than one disease state is also contemplated. In a more preferred embodiment, the IL-10 variant molecule is an EBV-IL10 variant, fusion protein, or chimeric protein thereof. In yet another embodiment, the IL-10 variant molecule, fusion protein, or chimeric protein thereof is used in combination therapy. For example, the IL-10 variant molecule, fusion protein, or chimeric protein thereof may be administered to a subject in combination with other therapies or treatments. In still other embodiments, a therapeutic amount of an IL-10 variant molecule, fusion protein, or chimeric protein thereof of the present application is administered to a subject suffering from a lipid-based disease, such as, but not limited to, elevated cholesterol levels.

[0020] In various embodiments, the IL-10 variant molecule or fusion protein thereof of the present application is delivered as an isolated and purified protein. Delivery may be in the form of a subcutaneous bolus injection or multiple microinjections into the dermis and subcutaneous tissue. In yet another embodiment, the IL-10 variant molecule may be delivered as a nucleic acid vector containing a sequence encoding the IL-10 variant, its fusion protein, or chimeric protein. The nucleic acid vector may be a plasmid or a viral particle carrying a viral vector. In one embodiment, the IL-10 variant molecule may be delivered to a subject by genetic medicine techniques commonly known to those skilled in the art.

[0021] In other embodiments, the IL-10 variant molecule may be administered as part of a combination treatment regimen. In one embodiment, the IL-10 variant molecule may be administered in combination with bispecific T-cell binding agents (BITES), which are immunotherapies currently available for the treatment of, for example, cancer, IBD, Crohn's disease, NAFLD, NASH, and autoimmune diseases.

[0022] In another embodiment, the nucleic acid vector is an adeno-associated virus (AAV) vector having one or more AAV inverted terminal repeat (ITR) sequence elements and regulatory elements for directing expression of the sequence encoding the IL-10 variant molecule in target cells; the AAV vector can be administered either as a plasmid ("naked" DNA) or packaged in an AAV particle. In another embodiment, the nucleic acid vector is a vaccinia virus vector. A variety of vaccinia virus vectors derived from different strains, such as the WR strain (ATCC VR-119), the Wyeth strain (ATCC VR-325), the Lederle-Chorioallantoic strain (ATCC VR-325), the CL strain (ATCC VR-117), and others, can be used to introduce the variant IL-10 molecules of the present application; all of these strains are available from the American Type Culture Collection (Manassas, Va.).

[0023] In another embodiment, any of the IL-10 variant molecules described herein, including but not limited to PEGylated IL-10 variant molecules, may be delivered to a subject by any method described herein or generally known in the art.

[0024] These and other embodiments of the present application will readily suggest themselves to those of ordinary skill in the art in light of the disclosure herein. The present invention provides, for example, the following items. (Item 1) Formulas (I to VII) IL10-L 1-X 1 -L 1 -X 2 -L 1 -IL10 (formula I), (Z) n -X 1 -L 2 -Y 2 -L 1 -IL10 (formula II), IL10-L 1 -Y 1 -L 2 -X 2 -(Z) n (Formula III), X 1 -L 2 -X 2 -L 1 -IL10 (Formula IV), IL10-L 1 -X 1 -L 2 -X 2 (Formula V), X 1 -L 1 -IL10 (Formula VI), IL10-L 1 -X2 (Formula VII), or any combination thereof A fusion protein of During the ceremony, "IL-10" is a monomer sequence selected from SEQ ID NOs: 1, 3, 14, 15, 16, 18, 19, 55, 57, or 59; "L 1 " is a linker of SEQ ID NO: 31 or 54, "L 2 " is the linker of SEQ ID NO: 30, "X 1" is a VH region obtained from a first antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as, but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin; SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola, "X 2 " is a VL region obtained from the same antibody as X1, "Y 1 " is a VH region obtained from a second antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as, but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MadCam, β7 integrin subunit; α4β7 integrin, α4 ​​integrin, SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola, "Y 2 " is a VL region obtained from the same antibody as Y1, X and Y are derived from the same or different antibodies, "Z" is a cytokine selected from IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferon-α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13; "n" is an integer selected from 0 to 2; Fusion proteins. (Item 2) Formulas II and III can form a fusion protein complex, and the IL-10 monomers derived from each of Formulas II and III can form a functional homodimeric IL-10 or The fusion protein according to item 1, which can form a variant. (Item 3) 3. The fusion protein of item 2, wherein Formula II is SEQ ID NO: 24, 26, 28, 41, 48, or 50. (Item 4) 3. The fusion protein of item 2, wherein Formula III is SEQ ID NO: 25, 27, 29, 42, 49, or 51. (Item 5) 3. The fusion protein of item 2, wherein the fusion protein complex is formed between SEQ ID NOs: 24 and 25, 26 and 27, 28 and 29, 41 and 42, 48 and 49, or 50 and 51. (Item 6) Item 1, wherein formulas IV and V are capable of forming a fusion protein complex, and IL-10 monomers derived from each of formulas IV and V are capable of forming a functional homodimeric IL-10 or a variant thereof. (Item 7) 7. The fusion protein of item 6, wherein Formula IV is SEQ ID NO: 35, 38, 46, 48, or 50. (Item 8) 7. The fusion protein of item 6, wherein Formula V is SEQ ID NO: 36, 39, 47, 49, or 51. (Item 9) 7. The fusion protein of item 6, wherein the fusion protein complex is formed between SEQ ID NOs: 35 and 36, 38 and 39, 46 and 47, 48 and 49, or 50 and 51. (Item 10) Item 1, wherein formulas VI and VII are capable of forming a fusion protein complex, and IL-10 monomers derived from each of formulas VI and VII are capable of forming a functional homodimeric IL-10 or a variant thereof. (Item 11) Item 2. The fusion protein according to item 1, wherein Formula I is SEQ ID NO: 33-34, 40, 43-44, 45, 52, or 53. (Item 12) 2. The fusion protein of item 1, wherein "n" is 1 or more and Z is IL-2, IL-7, IL-12, IL-15, or any combination thereof. (Item 13) Z is X 1 , Y 1 13. The fusion protein according to item 12, wherein the N-terminus of the fusion protein is conjugated to the N-terminus of either the nucleotide sequence of the fusion protein or the nucleotide sequence of the fusion protein. (Item 14) 10. A method for treating cancer, comprising administering to a patient in need thereof a composition comprising the fusion protein of item 1. (Item 15) Item 15. The method according to item 14, wherein the fusion protein is SEQ ID NO: 28 to 29, 35 to 36, 38 to 39, 46 to 47, 52, 53, 61, 63, 65, or 67. (Item 16) Item 16. The method of item 15, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID NOs: 28 and 29, between 35 and 36, between 38 and 39, or between 46 and 47. (Item 17) Item 14. The fusion protein comprises IL-10 consisting of DV07 of SEQ ID NO: 59. The method described below. (Item 18) 10. A method for treating an inflammatory disease, comprising administering to a patient in need thereof a composition comprising the fusion protein of item 1. (Item 19) Item 18. The method according to item 17, wherein the fusion protein is SEQ ID NO: 26 to 27, 41 to 42, 48, or 49. (Item 20) Item 19. The method of item 18, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID NOs: 26 and 27, 41 and 42, and 48 and 49. (Item 21) 19. The method of claim 18, wherein the composition comprises a fusion protein of SEQ ID NO: 37, 40, or 43. (Item 22) 19. The method of claim 18, wherein the fusion protein comprises IL-10 consisting of DV06 of SEQ ID NO: 57. (Item 23) 10. A method for treating a lipid-based disease, comprising administering to a patient in need thereof a composition comprising the fusion protein of item 1. (Item 24) Item 24. The method according to Item 23, wherein the fusion protein is SEQ ID NO: 24 to 25, 50, or 51. (Item 25) 25. The method of claim 24, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID NOs: 24 and 25 and between SEQ ID NOs: 50 and 51. (Item 26) 24. The method of claim 23, wherein the composition comprises a fusion protein of SEQ ID NO: 45. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 shows a ribbon diagram of the monomeric IL-10 molecule (Josephson et al., Immunity, 15, pp. 35-46). The red highlighting indicates the receptor contact boundary of Site I, and the green highlighting indicates the receptor contact boundary of Site II.

[0026] [Figure 2] Figures 2A-2C compare the ability of IL-10 and EBV IL-10 to activate monocytes / macrophages (Mθ) by testing IL-1β (Figure 2A) and TNFα (Figure 2B) cytokine production, as well as stimulation of T cells by testing IFNγ (Figure 2C) production in donor 1.

[0027] [Figure 3] Figures 3A-3C compare the ability of IL-10 and EBV IL-10 to activate monocytes / macrophages (Mθ) by testing IL-1β (Figure 3A) and TNFα (Figure 3B) cytokine production, as well as stimulation of T cells by testing IFNγ (Figure 3C) production in donor 1.

[0028] [Figure 4] 4A-B show the amount of IFNγ induction from T cells after stimulation with IL-10 or EBV IL-10.

[0029] [Figure 5] Figures 5A-5C show the effects of N-terminal 5 kDa mono- and di-PEGylated EBV-IL-10 on MC / 9 cell proliferation (Figure 5A), TNFα secretion by monocytes / macrophages (Mθ) in response to LPS (Figure 5B), and IFNγ secretion by T cells in response to T cell receptor stimulation (Figure 5C).

[0030] [Figure 6-1] 6A-6E show the specific amino acid sequences of various IL-10 variant molecules and fusion proteins, including EBV-IL-10. [Figure 6-2] 6A-6E show the specific amino acid sequences of various IL-10 variant molecules and fusion proteins, including EBV-IL-10. [Figure 6-3] 6A-6E show the specific amino acid sequences of various IL-10 variant molecules and fusion proteins, including EBV-IL-10. [Figure 6-4] 6A-6E show the specific amino acid sequences of various IL-10 variant molecules and fusion proteins, including EBV-IL-10. [Figure 6-5] 6A-6E show the specific amino acid sequences of various IL-10 variant molecules and fusion proteins, including EBV-IL-10.

[0031] [Figure 7] Figure 7 shows a schematic diagram of a fusion protein comprising an IL-10 or IL-10 variant molecule conjugated to a linker or spacer (in this case, an scFv). This representative example shows an IL-10 or IL-10 variant molecule conjugated to both the N- and C-terminus.

[0032] [Figure 8] Figures 8A-8C are schematic diagrams of various IL-10 variants, including variants created in EBV IL-10. Figure 8A (DV05) is an IL-10 variant containing a single point mutation (V31L V31L) at amino acid position 31 of SEQ ID NO: 3. Figure 8B (DV06) is an IL-10 variant containing a single point mutation (A75I A75I) at amino acid position 75 of SEQ ID NO: 3. Figure 8C (DV07) is an IL-10 variant containing two point mutations (V31L and A75I) at amino acid positions 31 and 75 of SEQ ID NO: 3.

[0033] Figure 8D assays the response of monocytes / macrophages to various forms of IL-10, including wild-type human IL-10, EBV-IL-10, DV05, DV06, and DV07. All forms, including IL-10 variants DV05, DV06, and DV07, suppress TNFα secretion in response to LPS. FIG. 8E assays T cell responses to various forms of IL-10, wild-type human IL-10, EBV IL-10, DV05, DV06, and DV07, and shows that not all forms induce IFN-gamma.

[0034] [Figure 9] 9A-9F are schematic diagrams of various configurations of IL-10 fusion protein / immunoconjugate / diabody constructs. Figures 9(a)-(c) depict fusion protein complexes (i.e., diabodies) in which each fusion protein comprises VH and VL regions derived from two different antibodies linked via carboxy- or amino-terminal linkers to a monomer of IL-10 (which may be substituted with an IL-10 variant molecule) with (a) a single mutation affecting IL-10 receptor binding, e.g., at amino acid 31, (b) a single mutation affecting IL-10 receptor binding, e.g., at amino acid 75, and (c) two mutations affecting IL-10 receptor binding, e.g., at amino acid positions 31 and 75. Figures 9(d)-(f) depict fusion protein complexes (i.e., minibodies) in which each fusion protein comprises a single VH or VL region derived from an antibody linked via a carboxy- or amino-terminal linker to a monomer of IL-10 (which may be substituted with an IL-10 variant molecule) with (d) a single mutation that affects IL-10 receptor binding, e.g., at amino acid position 31, (e) a single mutation that affects IL-10 receptor binding, e.g., at amino acid position 75, and (f) two mutations that affect IL-10 receptor binding, e.g., at amino acid positions 31 and 75.

[0035] [Figure 10]Figures 10(A)-10(F) are schematic diagrams of various configurations of IL-10 fusion protein / immunoconjugate / diabody constructs. Figures 10(a)-(c) depict single fusion proteins (i.e., minibodies) in which a monomer of IL-10 (which may be substituted with an IL-10 variant molecule) is linked to either a VH or VL from the same antibody via a carboxy- or amino-terminal linker, respectively, and the VH and VL are linked together. The IL-10 monomer contains (a) a single mutation at amino acid position 31 that affects IL-10 receptor binding, (b) a single mutation at amino acid position 75 that affects IL-10 receptor binding, and (c) two mutations at amino acids 31 and 75 that affect IL-10 receptor binding. 10(d)-(f) depict single fusion proteins in which monomers of IL-10 (which may be substituted with IL-10 variant molecules) are linked together, and each IL-10 monomer is further linked to a single VH or VL domain derived from an antibody via a carboxy- or amino-terminal linker. The IL-10 monomer contains (d) a single mutation at amino acid position 31 that affects IL-10 receptor binding, e.g., a single mutation at amino acid position 75 that affects IL-10 receptor binding, and (f) two mutations at amino acids 31 and 75 that affect IL-10 receptor binding.

[0036] [Figure 11]Figure 11 shows the reduction in tumor volume in vivo using a diabody containing the DV07 mutation. Formulation buffer (1x phosphate-buffered saline, "control") and the DV07 diabody (a fusion protein complex containing an EBV IL-10 variant with V31L V31L and A75I A75I in the mature protein, and variable domains from anti-CD3α and anti-EGFR, neither of whose VH / VL pairs binds to the mouse target) were administered as a single dose on day 3. Dose concentrations of 1 mg / kg and 0.4 mg / kg were tested for the DV07 diabody.

[0037] [Figure 12] Figure 12 shows in vitro T cell responses to published variants of IL-10 (diamonds), wild-type IL-10 (circles), and an alternative form of the DV07 diabody designated DHivDEbo:DV07 (diamonds, a fusion protein complex containing an EBV IL-10 variant with V31L and A75I mutations and variable domains derived from anti-HIV and anti-Ebola (neither of these VH / VL pairs binds to the mouse protein), where the diabody has the structure shown schematically in Figure 9(c)).

[0038] [Figure 13]FIG. 13A compares the suppression of TNFα induced by LPS exposure on monocytes / macrophages using various forms of the EBV IL-10 variant molecule with the V31L and A75I mutations in the form of a diabody. "wt" represents human IL-10, "EBV" represents EBV IL-10, "DCd3DEgfr:DV05" is a diabody comprising VH and VL regions derived from an anti-CD3 antibody and an anti-EGFR antibody linked to an EBV IL-10 variant containing the V31L mutation, "DCd3DEgfr:DV07" is a diabody comprising VH and VL regions derived from an anti-CD3 antibody and an anti-EGFR antibody linked to an EBV IL-10 variant containing both the V31L and A75I mutations, and "DHivDEbo:DV07" is a diabody comprising VH and VL regions derived from an anti-HIV antibody and an anti-Ebola antibody linked to an EBV IL-10 variant containing both the V31L and A75I mutations.

[0039] Figure 13B compares IFNγ secretion in T cells in a response assay using human IL-10 ("wt"), EBV IL-10 ("EBV"), an EBV IL-10 variant molecule with V31L and A75I mutations, and various diabody forms containing VH and VL regions derived from different antibodies. Form DHivDEgfr:DV07 is an EBV IL-10 variant diabody with V31L and A75I substitutions containing variable regions derived from anti-HIV and anti-EGFR. Form DHivDEbo:DV07 is an EBV IL-10 variant diabody with both V31L and A75I mutations containing variable regions derived from anti-HIV and anti-Ebola.

[0040] [Figure 14]Figures 14A-14B compare two forms of EBV IL-10 variant diabodies, DH:DV07 and DHDE:DV07, in monocytes / macrophages (Figure 14A) and T cells (Figure 14B) isolated from two donors. Form DHDV07 is an EBV IL-10 variant diabody with V31L and A75I substitutions that contains variable regions derived from anti-HIV and anti-EGFR antibodies. Form DHDE:DV07 is an EBV IL-10 variant diabody with V31L and A75I substitutions that contains variable regions derived from anti-HIV and anti-Ebola antibodies. Figure 14A compares the suppression of LPS-induced TNFα in isolated monocytes / macrophages using human IL-10 ("wt"), DH:DV07, and DHDE:DV07. FIG. 14B compares IFNγ secretion in isolated T cells in response to human IL-10 (“wt”), EBV IL-10, DH:DV07, and DHDE:DV07.

[0041] [Figure 15] Figure 15 is a direct comparison of various forms of EBV-10 variant diabody forms in MC / 9 mast cells. The assay compares human IL-10, EBV IL-10, D:DV05 (EBV IL-10 with a V31L mutation, containing variable regions derived from anti-CD3α and anti-EGFR), D:DV06 (EBV IL-10 with an A75I mutation, containing variable regions derived from anti-CD3α and anti-EGFR), D:DV07 (EBV IL-10 variant diabody with a V31L and A75I substitution, containing variable regions derived from anti-CD3α and anti-EGFR), and DhivDEbo:DV07 (EBV IL-10 variant diabody with a V31L and A75I substitution, containing variable regions derived from anti-HIV and anti-Ebola).

[0042] [Figure 16]Figures 16A-16C show results from an in vivo tumor study using D:DV07 (containing variable regions derived from anti-CD3α and anti-EGFR, with V31L and A75I mutations). In vivo tumor volumes were assessed following administration of dosing formulation buffer ("control"), 0.4 mg / kg three times per week (q3w), 0.2 mg / kg three times per week (q3w), 0.2 mg / kg with two days off (qd), and 0.1 mg / kg with two days off (qd).

[0043] [Figure 17] Figures 17A-17B show results from an in vivo study of two IL-10 variant fusion protein formats, large and small, corresponding to the schematic diagrams in Figures 9C and 9F, respectively. The IL-10 variants contain the V31L and A75I mutations. These results examined the effect of IL-10 fusion proteins without targeting capabilities on tumor size reduction. The VH and VL regions derived from the fusion proteins are non-targeting sequences derived from either anti-HIV and anti-Ebola (large) or anti-Ebola (small). Figure 17A shows a dosing study of the non-targeting mini-format compared to pegylated IL-10 (0.75 mg / kg daily), administered for 5 days on, 2 days off. Figure 17B is a dosing study of non-targeted mini (1 mg / kg, 0.5 mg / kg, 0.25 mg / kg) and large (0.2 mg / kg) formats using three times weekly dosing compared to pegylated recombinant human IL-10 (0.75 mg / kg, daily).

[0044] [Figure 18]Figures 18A-18C show results from an in vivo study of two IL-10 variant fusion protein formats, large and small, corresponding to the schematic diagrams in Figures 9C and 9F, respectively. These results examined the effect of targeting-capable IL-10 fusion proteins on tumor size reduction. The IL-10 variants contain the V31L and A75I mutations. In the large format, one set of VH and VL regions from the fusion protein is derived from an anti-EGFR antibody, and the other set of VH and VL is derived from an anti-Ebola antibody. The small format contains VH and VL derived solely from an anti-EGFR antibody. Figure 18A shows results from a daily dosing study of the large (0.25 mg / kg), small (0.25 mg / kg), and non-targeted IL-10 fusion protein small (0.25 mg / kg) formats compared with pegylated recombinant human IL-10 (0.75 mg / kg). Figure 18B shows results from a three times weekly dosing study of large format targeted IL-10 fusion protein (1 mg / kg, 0.25 mg / kg, and 0.25 mg / kg qd) compared with large format non-targeted IL-10 fusion protein (DhDe:DV07, 0.2 mg / kg) and pegylated IL-10 (0.75 mg / kg qd). Figure 18C shows results from a three times weekly dosing study of small format targeted IL-10 fusion protein (1 mg / kg, 0.25 mg / kg) compared with small format non-targeted IL-10 fusion protein (Debo:DV07, 1 mg / kg) and pegylated IL-10 (0.75 mg / kg qd).

[0045] [Figure 19] 19A-19B show the results obtained from an in vivo cholesterol study using a diabody with an EBV IL-10 variant with a V31L mutation, containing variable regions derived from anti-CD3α and anti-EGFR.

[0046] [Figure 20]Figures 20A-20B show results from an in vitro comparative study of two IL-10 variant fusion proteins on macrophages and T cells. The assay tests the in vitro efficacy of two forms of the DV06 fusion protein compared to human IL-10. Figure 20A shows a monocyte / macrophage assay using DhivDebo:DV06 (SEQ ID NOs: 26 and 27) and DmadcamDEbo:DV06 (SEQ ID NOs: 41 and 42). Figure 20B shows a T cell response assay, as measured by IFN-gamma, using DhivDebo:DV06 (SEQ ID NOs: 26 and 27) and DmadcamDEbo:DV06 (SEQ ID NOs: 41 and 42).

[0047] [Figure 21-1] Figures 21A-21D are EBV IL-10 amino acid sequences. Figure 21A is EBV IL-10. Figure 21B is DV05, which contains a V31L substitution. Figure 21C is DV06, which contains an A75I substitution. Figure 21D is DV07, which contains both V31L and A75I substitutions. [Figure 21-2] Figures 21A-21D are EBV IL-10 amino acid sequences. Figure 21A is EBV IL-10. Figure 21B is DV05, which contains a V31L substitution. Figure 21C is DV06, which contains an A75I substitution. Figure 21D is DV07, which contains both V31L and A75I substitutions. [Figure 21-3] Figures 21A-21D are EBV IL-10 amino acid sequences. Figure 21A is EBV IL-10. Figure 21B is DV05, which contains a V31L substitution. Figure 21C is DV06, which contains an A75I substitution. Figure 21D is DV07, which contains both V31L and A75I substitutions. [Figure 21-4] Figures 21A-21D are EBV IL-10 amino acid sequences. Figure 21A is EBV IL-10. Figure 21B is DV05, which contains a V31L substitution. Figure 21C is DV06, which contains an A75I substitution. Figure 21D is DV07, which contains both V31L and A75I substitutions. DETAILED DESCRIPTION OF THE INVENTION

[0048] Detailed Description of Various Preferred Embodiments Before describing the applications of various embodiments in detail, it is to be understood that the present application is not limited to particular formulations or process parameters, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing various embodiments only, and is not intended to be limiting.

[0049] Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of various described embodiments, the preferred materials and methods are described herein.

[0050] Unless otherwise indicated, the embodiments described herein employ conventional methods and techniques of molecular biology, biochemistry, pharmacology, chemistry, and immunology that are well known to those skilled in the art. Many of the general techniques for designing and producing IL-10 variants, including but not limited to human, CMV, and / or EBV forms of IL-10, as well as assays for testing IL-10 variants, are readily available and well-known in the art, and are well-described in detail. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods in Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell eds., Blackwell Scientific Publications); and A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition). N-terminal aldehyde-based P EGylation chemistry is also well known in the art.

[0051] The following terms are used to describe the various embodiments discussed herein and are intended to be defined as set forth below.

[0052] As used herein in describing various embodiments, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0053] The term "about" refers to a deviation of 0.0001 to 5% from a given number or range of numbers. In one embodiment, the term "about" refers to a deviation of 1 to 10% from a given number or range of numbers. In one embodiment, the term "about" refers to a deviation of up to 25% from a given number or range of numbers. In more specific embodiments, the term "about" refers to a difference of 1 to 25% in terms of nucleotide sequence homology or amino acid sequence homology when compared to a wild-type sequence.

[0054] The terms "interleukin-10" or "IL-10" refer to a protein comprising two subunits non-covalently joined to form a homodimer, where IL-10 is an intercalated dimer composed of two six-helix bundles (helices A-F). As used herein, unless otherwise indicated, "interleukin-10" and "IL-10" can refer to human IL-10 ("hIL-10", Genbank Accession No. NP_000563, or U.S. Patent No. 6,217,857) protein (SEQ ID NO: 1) or nucleic acid (SEQ ID NO: 2), murine IL-10 ("mIL-10", Genbank Accession No. M37897, or U.S. Patent No. 6,217,857) protein (SEQ ID NO: 7) or nucleic acid (SEQ ID NO: 8), or viral IL-10 ("vIL-10"). Viral IL-10 homologs can be derived from EBV or CMV (Genbank accession numbers NC_007605 and DQ367962, respectively). The term EBV-IL10 refers to the EBV homolog of the IL-10 protein (SEQ ID NO: 3) or nucleic acid (SEQ ID NO: 4). The term CMV-IL10 refers to the CMV homolog of the IL-10 protein (SEQ ID NO: 5) or nucleic acid (SEQ ID NO: 6). The term monomeric IL-10, as used herein, refers to individual subunits of IL-10 or variant IL-10 that, when non-covalently joined, form IL-10 or variant IL-10 homodimers. The terms "wild-type," "wt," and "native" are used interchangeably herein to refer to the sequence of a protein (e.g., IL-10, CMV-IL10, or EBV-IL10) that is commonly found in nature in the species from which the particular IL-10 in question originates. For example, the term "wild-type" or "native" EBV-IL10 would therefore correspond to the amino acid sequence most commonly found in nature.

[0055] The terms "derive," "derived," "derive from," or "derived from" are used herein to identify the origin of a molecule, e.g., a viral form of an IL-10 molecule, and are not meant to limit the manner in which the molecule is prepared, manufactured, produced, or made. This would include methods such as, but not limited to, chemical or recombinant means.

[0056] The term "derivative" is intended to include any suitable modified form of the reference molecule of interest or its analog, such as sulfation, acetylation, glycosylation, phosphorylation, polymer conjugation (e.g., with polyethylene glycol), hesylation, or addition of other foreign moieties, so long as the desired biological activity of the reference molecule or variant (e.g., anti-inflammatory and / or lack of T cell stimulation) is retained.

[0057] The terms "variant," "analog," and "mutein" refer to a biologically active derivative of a reference molecule that retains a desired activity, such as, for example, anti-inflammatory activity. Generally, the terms "variant," "variants," "analog," and "mutein," when referring to a polypeptide, refer to one or more compounds having a native polypeptide sequence and structure that have one or more amino acid additions, substitutions (conservative in nature), and / or deletions compared to the native molecule. Thus, the terms "IL-10 variant," "variant IL-10," "IL-10 variant molecule," and grammatical variations and plural forms thereof, are all intended to be equivalent terms referring to an IL-10 amino acid (or nucleic acid) sequence that differs from wild-type IL-10 by anywhere from 1 to 25% sequence identity or homology. Thus, for example, an EBV IL-10 variant molecule differs from wild-type EBV IL-10 by having one or more amino acid (or amino acid-encoding nucleotide) additions, substitutions, and / or deletions. Thus, in one aspect, an EBV IL-10 variant differs from the wild-type sequence of SEQ ID NO: 3 by having about a 1% to 25% difference in sequence homology, which corresponds to about 1 to 42 amino acid differences.

[0058] The term "fusion protein" refers to the combination or conjugation of two or more proteins or polypeptides, resulting in a novel protein configuration not normally found in nature. Fusion proteins are the result of the covalent linkage of two or more proteins or polypeptides. The two or more proteins comprising a fusion protein can be arranged in any configuration from amino to carboxy terminus. Thus, for example, the carboxy terminus of one protein can be covalently linked to either the carboxy or amino terminus of another protein. An exemplary fusion protein can include a monomeric IL-10 or monomeric variant IL-10 molecule combined with one or more antibody variable domains. Fusion proteins can also dimerize or associate with other fusion proteins of the same type, resulting in a fusion protein complex. Fusion protein complex formation can, in some cases, activate or increase the functionality of the fusion protein compared to the uncomplexed fusion protein. For example, a monomeric IL-10 or monomeric variant IL-10 molecule having one or more antibody variable domains may have limited or reduced ability to bind to the IL-10 receptor; however, when the fusion protein is complexed, the monomeric IL-10 or variant IL-10 molecule homodimerizes and the variable domains associate into a functional antibody.

[0059] "Functional variants" are IL-10 variant molecules that contain modifications (e.g., additions, substitutions, and / or deletions) that do not destroy the biological activity of the reference molecule. These variants may be "homologous" to the reference molecule as defined below. Generally, the amino acid sequence of such analogs will have a high degree of sequence identity to the reference sequence when the two sequences are aligned, e.g., greater than 50%, typically greater than 60%-70%, and even more specifically, 80%-85% or more, e.g., at least 90%-95% or more amino acid sequence identity. Often, analogs will contain the same number of amino acids but will contain substitutions. Functional variants will retain enhanced, attenuated, or substantially the same biological activity as the native molecule. Specifically, the term "variant" IL-10 molecule is interchangeable with the term "engineered" IL-10 molecule or IL-10 variant molecule or IL-10 variant and refers to an IL-10 molecule or protein that contains one or both modifications to the IL-10 receptor-binding domain and / or regions involved in forming the interdomain angle or interhomodimer angle in the IL-10 molecule or protein. A variant IL-10 "fusion protein" or "diabody" or "fusion" generally refers to the formation of a fusion protein (or fusion protein complex) comprising variant IL-10 (in either monomeric or homodimeric form) and at least one other protein. As used herein, variant IL-10 "or fusion protein thereof" may be used throughout the specification to describe such variant IL-10 fusion proteins.

[0060] An "analog" or "analogs" may include substitutions that are conservative in nature. For example, conservative substitutions may include, but are not limited to, types of substitutions such as: (1) an acidic substitution between aspartic acid and glutamic acid; (2) a basic substitution between any one of lysine, arginine, or histidine; (3) a nonpolar substitution between any one of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; and (4) an uncharged polar substitution between any one of glycine, asparagine, glutamine, cysteine, serine, threonine, or tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. It is also possible to make single substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar conservative substitutions of one amino acid with a structurally related amino acid, so long as the specific biological activity desired remains intact. For example, a polypeptide of interest can contain up to about 1-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 or any integer number between 1-50, conservative or non-conservative amino acid substitutions, so long as the desired function of the molecule remains intact. One of skill in the art can readily determine regions of a molecule of interest that can tolerate alterations well known in the art.

[0061] "Mutein" further includes compounds containing only amino and / or imino molecules, polypeptides containing one or more amino acid analogs (including, e.g., unnatural amino acids, etc.), polypeptides with substituted linkages, and polypeptides having one or more amino acid-like molecules, including, but not limited to, other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic) cyclized branched molecules, etc. Preferably, the analog or mutein will retain some biological activity that is enhanced, attenuated, or substantially the same as the native molecule. Methods for making polypeptide analogs and muteins are well known in the art.

[0062] The terms "homolog," "homology," "homologous," or "substantially homologous" refer to the percent identity between at least two polynucleotide sequences or at least two polypeptide sequences. Sequences are homologous to one another if they exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a given length of the molecules.

[0063] The term "sequence identity" refers to the exact correspondence between nucleotides or amino acids. Sequence identity can range from 100% sequence identity to 50% sequence identity. Percent sequence identity can be determined using a variety of methods, including, but not limited to, direct comparison of sequence information between two molecules (a reference sequence and a sequence with an unknown percent identity to the reference sequence) by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. Readily available computer programs can be used to assist in determining percent identity.

[0064] The term "fragment" is intended to include molecules that are portions of a full-length amino acid or polynucleotide sequence and / or structure. A fragment of a polypeptide can include, for example, a C-terminal deletion, an N-terminal deletion, and / or an internal deletion of the native polypeptide. An active or functional fragment of a particular protein generally includes at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer number between 5 amino acids and the full-length sequence, provided that the fragment in question retains biological activity, e.g., anti-inflammatory activity. In the context of antibodies, an antibody fragment refers to a portion of an intact antibody that includes the antigen-binding site or variable region (heavy and / or light chain regions) of the intact antibody. These include, for example, Fab, Fab', Fab'-SH, (Fab')2, Fv fragments, diabodies, single-chain Fvs (ScFvs), single-chain polypeptides having one light chain variable domain, and fragments having three CDRs of the light chain variable domain or the heavy chain variable domain.

[0065] The term "substantially purified" generally refers to the isolation of a substance such that the substance constitutes the majority percentage of the sample in which it is present. A substantially purified component will constitute 50%, preferably 80%-85%, and more preferably 90-95% of a sample. Similarly, the term "isolated," when referring to a polypeptide or polynucleotide, means that the referenced molecule is separated and distinct from the whole organism in which it is naturally found, or exists in the substantial absence of other biological macromolecules of the same type.

[0066] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, including, but not limited to, murines, rodents, simians, humans, farm animals, sport animals, and certain pets.

[0067] The term "administering" includes routes of administration that allow the active ingredients of the present application to perform their intended function.

[0068] A "therapeutically effective amount," e.g., in the context of administration of an EBV-IL-10 variant or fusion protein thereof described herein, refers to an amount of EBV-IL-10 variant or fusion protein thereof that is sufficient to promote a particular biological activity, including, e.g., suppression of myeloid cell function, enhancement of Kupffer cell activity, and / or CD8 + Lack of any effect on T cells or CD8 + It can include enhancing T cell activity, and blocking the mast cell upregulation of Fc receptor or preventing degranulation.Therefore, "effective amount" alleviates or prevents the symptoms or signs of medical condition.Effective amount also means the amount that is sufficient to enable or facilitate diagnosis.

[0069] The term "treat" or "treatment" refers to a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the underlying cause of the disease or condition itself, not just the symptoms. Treatment can be any reduction from original levels, and can be, but is not limited to, the complete elimination of the disease, condition, or symptoms of the disease or condition.

[0070] A "chemotherapeutic" agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa, cyclosphosphamide, (CYTOXAN™), alkyl sulfonates such as busulfan, improsulfan, and piposulfan, aziridines such as benzodopa, carboquone, mesuredopa, and uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphatamide ethyleneimines and methylamelamines, including iethylenethiophosphaoramide, and trimethylolomelamime; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride;hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, antibiotics such as aclanomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, Carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin (potfiromycin), puromycin, quelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate, purine analogs such as fludarabine, 6-mercaptopurine, thiamprinium thioguanine, pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, anti-adrenals such as aminoglutethimide, mitotane, trilostane, folic acid replenishers such as florinic acid, aceglatone, aldophosphamideglycoside), aminolevulinic acid, amsacrine, bestrabucil, bisantrene, edatrexate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamt, pirarubicin, podophyllinic acid acid), 2-ethylhydrazide, procarbazine, PSK®, razoxane, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (Ara-C), cyclophosphamide, thiotepa, taxoids such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (Taxotere™, Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, Xeloda® Roche Switzerland, ibandronate, CPT11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoic acid, esperamicin, capecitabine, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Also included within this definition are antihormonal agents that act to regulate or inhibit the action of hormones on tumors, e.g., antiestrogens, including, by way of example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston), and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0071] The terms "conjugate," "conjugated," "conjugation," or "conjugating," as used in this application, refer to the linking together of two or more portions of a molecule. The linkage occurs by a covalent bond (e.g., a peptide bond). IL-10 variant proteins

[0072] The IL-10 variant molecules of the present application include modifications to any form of IL-10. These modifications to the IL-10 molecule include the addition, deletion, and / or substitution of one or more amino acids in regions and / or domains involved in IL-10 receptor binding and / or those involved in conferring interdomain or interhomodimer angles in the IL-10 molecule. Exemplary IL-10 sequences that may be useful in constructing variant IL-10 molecules of the present application include those derived from Epstein-Barr virus ("EBV"; see, e.g., Moore et al., Science (1990) 248:1230-1234; Hsu et al., Science (1990) 250:830-832; Suzuki et al., J. Exp. Med. (1995) 182:477-486), cytomegalovirus ("CMV"; see, e.g., Lockridge et al., Science (1990) 248:1230-1234; Hsu et al., Science (1990) 250:830-832; Suzuki et al., J. Exp. Med. (1995) 182:477-486), and the like. al., Virol. (2000) 268:272-280; Kotenko et al., Proc. Natl. Acad. Sci. USA (2000) 97:1695-1700), and equine herpesvirus (see, e.g., Rode et al., Virus Genes (1993) 7:111-116), OrF virus (See, e.g., Imlach et al., J. Gen. Virol. (2002) 83:1049-1058 and Fleming et al., Virus Genes (2000) 21:85-95) Other representative IL-10 sequences include, but are not limited to, those listed under NCBI accession numbers NM010548, AF307012, M37897, M84340 (mouse sequence), U38200 (horse sequence), U39569, AF060520 (feline sequence), U00799 (bovine sequence), U11421, Z29362 (ovine sequence), L26031, L26029 (macaque sequence), AF294758 ( Examples of sequences described in U33843 (dog), AF088887, AF068058 (rabbit sequences), AF012909, AF120030 (woodchuck sequences), AF026277 (opossum), AF097510 (guinea pig), U11767 (deer), L37781 (gerbil), AB107649 (llama and camel).

[0073] In one embodiment, the IL-10 variant molecules described herein are obtained by modifying the human (SEQ ID NO: 1), CMV (SEQ ID NO: 5), EBV (SEQ ID NO: 3) IL-10 sequence, or the mouse (SEQ ID NO: 7) wild-type protein. Representative examples of various IL-10 variant molecules are provided in SEQ ID NOs: 9-23.

[0074] Modifications to wild-type IL-10 include the addition, deletion, and / or substitution of one or more amino acids in regions involved in (i) IL-10 receptor binding and / or (ii) the formation of interdomain or interhomodimer angles of the IL-10 molecule. Variant IL-10 molecule: IL-10 receptor binding domain

[0075] Regions involved in receptor binding include any amino acid moieties located within the region directly involved in or responsible for binding of IL-10 to IL-10 receptor 1 (IL10R1) and / or IL-10 receptor 2 (IL10R2). These regions may include, for example, discontinuous portions of the IL-10 molecule previously discussed and mapped in the art (see, e.g., Yoon 2005; Josephson 2001). Modifications to any region involved in forming contact or breakpoints associated with binding of IL-10 to the IL-10 receptor, such as, but not limited to, helix A, helix F, and the AB loop, are contemplated in the present application. In a preferred embodiment, modifications (e.g., additions, deletions, and / or substitutions) to the receptor-binding domain include amino acids 31 and / or 75 of SEQ ID NO:3. In particularly preferred embodiments, the modification comprises a substitution of valine at position 31 of SEQ ID NO: 3 with leucine (V31L, referred to herein as "DV05"), a substitution of alanine at position 75 of SEQ ID NO: 3 with isoleucine (A75I, referred to herein as "DV06"), or substitution of both V31L and A75I in SEQ ID NO: 3 (referred to herein as "DV07"). In one aspect, DV05 is SEQ ID NO: 55, DV06 is SEQ ID NO: 57, and DV07 is SEQ ID NO: 59.

[0076] In one embodiment, the modification to the receptor-binding domain comprises any one or more of the Site Ia and / or Site Ib interface contact points discussed by Josephson et al. (Immunity, 2001, 15, pp. 35-46, Figure 1). In one embodiment of the present application, the Site Ia interface contact point comprises one or more amino acids located at the bend in helix F and in the AB loop. In another embodiment, the Site Ib interface contact point comprises one or more amino acids located at the N-terminus of helix A and the C-terminus of helix F. In another embodiment, any one or more amino acids located in IL-10 involved in receptor binding comprise any one or more of 1 to 10 amino acids in helix A and 1 to 7 amino acids in the AB loop. In another embodiment, the receptor binding region can include one or more modifications of the following amino acids or 1 to 10 amino acids centered thereon, where the amino acids are Glu-142, Lys-138, Asp-144, Gln-38, Ser-141, Asp-44, Gln-42, Gln-38, Arg-27, Glu-151, Arg-24, Pro-20, Ile-158, or any combination thereof.

[0077] In one aspect, the modifications to the receptor-binding domain include any one or more of the Site IIa and / or Site IIb interface contact points discussed by Josephson et al. (Immunity, 2001, 15, pp. 35-46, Figure 1). In one embodiment of the present application, the Site IIa interface contact point includes one or more amino acids located in the DE loop. In another embodiment, the receptor-binding region may include one or more modifications of the following amino acids, or 1 to 10 amino acids centered thereon, where the amino acids are Ser-11, Thr-13, Asn-18, Arg-104, Arg-107, or any combination thereof. In one embodiment, the variant IL-10 molecule includes 1 to 100 (or any integer therein) amino acid additions, deletions, and / or substitutions that affect the receptor-binding domain, such additions, deletions, and / or substitutions either increasing or decreasing the binding affinity of the variant IL-10 molecule for the IL-10 receptor. Variant IL-10 molecules: Altered interdomain angles

[0078] The region involved in the formation of the interdomain (interhomodimer, which are used interchangeably) angle of the IL-10 molecule includes any amino acid moiety located within the region directly involved in or responsible for the formation of a specific interdomain angle of the IL-10 homodimer. Wild-type IL-10 forms an "L-shaped" dimer when two monomer units of IL-10 interconnect in an antiparallel manner. The resulting interdomain angles of human IL-10 and EBV-IL10 have been reported to be approximately 89 degrees and 97 degrees, respectively. To modulate IL-10 receptor signaling, in one embodiment, the present application explores modifying amino acids within the DE loop, which is part of helix D or helix E involved in the formation of the L-shaped dimer, in each monomer involved in the formation of the interdomain angle. In another embodiment, the region involved in the interdomain angle includes a linker region of approximately 12 amino acids located between helix D and helix E of the IL-10 protein. The modifications by addition, deletion, or substitution result in a restricted or relaxed IL-10 interdomain angle when compared to either human IL-10 or EBV-IL10. The monomeric IL-10 molecule is modified to result in a restricted / tight / closed or relaxed / loose / open IL-10 interdomain angle upon homodimerization, producing a variant IL-10 molecule with an altered interdomain angle that binds to and modulates its cognate receptor (IL-10 receptor). In another embodiment, the substitution comprises introducing a proline into an amino acid segment located between the D and E helices and / or between the C and D helices of EBV-IL10.

[0079] Thus, in certain embodiments, a variant IL-10 molecule will contain one or more additions, deletions, and / or substitutions that result in an altered intermolecular angle or altered interdomain angle when compared to wild-type IL-10. The altered intermolecular angle or altered interdomain angle can result in the variant IL-10 molecule dimerizing with the same or a different variant IL-10 molecule and binding to the IL-10 receptor at a different binding angle when compared to the wild-type IL-10 molecule. The different binding angle of the variant IL-10 molecule results in the ability to modulate or "tune" IL-10 receptor signaling to either activate or suppress inflammation and / or immune responses. In a preferred embodiment, the variant IL-10 molecule is EBV-IL10. In another preferred embodiment, the variant IL-10 molecule uses the EBV-IL10 molecule as a reference for modification. In yet another embodiment, the variant IL-10 molecule is a hybrid molecule that obtains portions and domains from other IL-10 molecules (such as, but not limited to, human IL-10, murine IL-10, and / or CMV-IL10).

[0080] In another preferred embodiment, the variant IL-10 molecule provides a relaxed interdomain angle that suppresses inflammatory cell (myeloid lineage cell) responses and does not induce activation of lymphoid cells, such as T cells. When combined with modifications to the receptor-binding domain, preferably modifications that result in reduced or unchanged receptor affinity, variant IL-10 molecules with a relaxed interdomain angle will be effective in suppressing myeloid cell (monocytes, macrophages, neutrophils, granulocytes, mast cells, Kupffer cells) cytokine secretion in response to pro-inflammatory stimuli. This configuration of the variant IL-10 molecule is useful for treating inflammatory diseases such as, but not limited to, IBD, Crohn's disease, psoriasis, rheumatoid arthritis, NAFLD, and NASH.

[0081] In another preferred embodiment, the variant IL-10 molecule provides a restricted interdomain angle that enhances activation of immune cells, e.g., T cells. When combined with modifications to the receptor binding domain, preferably modifications that result in high receptor affinity, variant IL-10 molecules with restricted interdomain angles enhance activation of immune cells, e.g., CD8 + This configuration of the variant IL-10 molecule would be useful in treating a variety of solid and hematopoietic cancers, including, for example, metastatic cancers, and would be effective in enhancing the elimination of T cells, NK cells, and Kupffer cells.

[0082] The regions involved in forming the interdomain angle can be continuous or discontinuous portions located within the IL-10 molecule. In one embodiment, the interdomain angle of the IL-10 variant molecule has an angle variation of 1 to 25 degrees, in another preferred embodiment the angle variation is 1 to 10 degrees, in a more preferred embodiment the angle variation is 1 to 5 degrees, and in a most preferred embodiment the angle variation is less than 5 degrees. In one embodiment, the variant IL-10 molecule will contain 1 to 100 (or any integer number therein) amino acid additions, deletions, and / or substitutions that affect the interdomain angle.

[0083] In one embodiment of the present application, variant IL-10 molecules will be designed and generated with the aid of computer-based modeling to predict the region or regions most involved in IL-10 receptor binding and / or interdomain angles. Computer-based modeling will help provide a fast and efficient means of predicting regions that would most benefit from modifications to the receptor binding domain and / or interdomain angles.

[0084] In another embodiment, the molecules of the present application include derivatives of variant IL-10 molecules, which may include modifications to the variant molecule to include entities that increase the size, half-life, and bioavailability of the variant molecule. Variant IL-10 molecules: PEG-modified

[0085] In one embodiment, the variant IL-10 molecule may include the attachment of polyethylene glycol (PEG). The PEGylated IL-10 variant may include the attachment of at least one PEG molecule. Without being bound to any particular theory, the attachment of PEG to the IL-10 variant may promote destabilization of the IL-10 variant on the receptor to protect it from proteolysis, reduce immunogenicity, and maintain its suppressive effect on myeloid cells, preventing T cell activation.

[0086] In its most common form, PEG is a linear or branched polyether terminated with hydroxyl groups and has the following general structure: HO-(CH2CH2O) n -CH2CH2-OH

[0087] The method of linking PEG to the variant IL-10 molecule of the present application follows techniques / protocols already established in the art. For example, conjugation or linking of PEG requires activating PEG by preparing a derivative of PEG with a functional group at one or both ends. The most common route for PEG conjugation of proteins is to activate PEG with a functional group suitable for reaction with lysine and the N-terminal amino acid group. In particular, the most common reactive group involved in linking PEG to polypeptides is the alpha or epsilon amino group of lysine.

[0088] Reaction of a PEGylated linker with a variant IL-10 molecule results in attachment of PEG moieties primarily to the following sites: the alpha amino group at the N-terminus of the protein, the epsilon amino groups on the side chains of lysine residues, and the imidazole groups on the side chains of histidine residues. In some embodiments, the variant IL-10 molecule is a recombinant protein with a single alpha amino group and several epsilon amino and imidazole groups, and multiple positional isomers can be generated depending on the chemical nature of the linker.

[0089] Two widely used first-generation activated monomethoxy PEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG, e.g., Zalipsky, et al. (1992) Biotechnol. Appl. Biochem 15:100-114 and Miron and Wilcheck (1993) Bioconjug. Chem. 4:568-569) and benzotriazole carbonates. PEG (BTC-PEG, see, e.g., U.S. Patent No. 5,650,234 to Dolence et al.), which reacts preferentially with lysine residues to form carbamate linkages, but is also known to react with histidine and tyrosine residues. The linkage to histidine residues on IFNα is known to be a hydrolytically unstable imidazole carbamate linkage (see, e.g., Lee and McNemar, U.S. Patent No. 5,985,263). (See, e.g., pp. 111-114, 2003, which are incorporated herein by reference in their entireties).

[0090] Second-generation PEGylation technology is designed to avoid these unstable linkages and the lack of selectivity in residue reactivity. The use of PEG-aldehyde linkers targets a single site on the N-terminus of a polypeptide and / or protein subunit through reductive amination. IL-10 can be PEGylated using different types of linkers and pH to obtain various forms of PEGylated molecules (see, e.g., U.S. Patent No. 5,252,714; U.S. Patent No. 5,643,575; U.S. Patent No. 5,919,455; U.S. Patent No. 5,932,462; U.S. Patent No. 5,985,263; U.S. Patent No. 7,052,686, all of which are incorporated by reference in their entirety). IL-10 mimetic molecules

[0091] In another embodiment, the present application encompasses mimetic molecules that reflect the biological function of IL-10 variant molecules. These mimetics include, but are not limited to, peptides, small molecules, modified hormones, and antibodies that have the same or substantially the same structure and / or function as those produced by the variant IL-10 molecules. IL-10 mimetic molecules that can form the basis for modification to replicate or reflect IL-10 variant molecules include those described in U.S. Publication Nos. US20080139478, US20120238505, and / or US20150218222, all of which are incorporated by reference in their entireties. IL-10 hybrid molecules and IL-10 fusion proteins

[0092] In another embodiment, the present application includes IL-10 variant molecules that are hybrid molecules composed of portions obtained from human IL-10, EBV-IL10, and / or CMV-IL10. For example, different domains within each of human IL-10, EBV-IL10, and / or CMV-IL10 can be combined together to create a hybrid molecule, such that the combination utilizes all or part of the domains involved in the receptor binding domain and / or interdomain angle in IL-10.

[0093] In one other embodiment, the variant IL-10 molecule is part of an engineered fusion protein. The linker or spacer can be a random amino acid sequence (e.g., SSGGGGGS (SEQ ID NO: 30, GGGGSGGGGSGGGGS (SEQ ID NO: 31), or SSGGGGSGGGGSGGGGS (SEQ ID NO: 54)), the constant region of an antibody, scFv, or diabody. The constant region can be derived from, but is not limited to, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE. The linker or spacer is preferably heavy chain constant (CH) region 1, CH2, or CH3. In a more preferred embodiment, the linker of the spacer is the random amino acid sequence of SEQ ID NO: 30 and / or 31. In another aspect, the linker or spacer can further comprise at least two interchain disulfide bonds.

[0094] The fusion protein may also include at least one monomer of an IL-10 or IL-10 variant molecule conjugated to the N-terminus, C-terminus, or both of the fusion protein. In another embodiment, a fusion protein comprising IL-10 or an IL-10 variant may also include at least one cytokine conjugated to the opposite end from the IL-10 or variant IL-10, including IL-2, IL-7, IL-15, IL-26, IL-27, IL-28, IL-29, IL-10, an IL-10 variant molecule, IFN-alpha, TGF-beta, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or any combination thereof. In some preferred embodiments, the fusion protein comprises two monomeric forms of IL-10 or IL-10 variant molecules conjugated to the N-terminus of the fusion protein and two IL-10 or IL-10 variant molecules conjugated to the C-terminus of the fusion protein; the fusion protein comprises two monomeric forms of IL-10 or IL-10 variant molecules conjugated to the N-terminus of the fusion protein and at least one IL-2 molecule conjugated to the C-terminus of the fusion protein; and the fusion protein comprises two IL-10 or IL-10 variant molecules conjugated to the N-terminus of the fusion protein and at least one IL-15 molecule conjugated to the C-terminus of the fusion protein. In another embodiment, the C-terminus of the fusion protein may have at least two different cytokines selected from IL-2, IL-7, IL-15, IL-26, IL-27, IL-28, IL-29, IL-10, an IL-10 variant molecule, IFN-alpha, TGF-beta, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13.

[0095] In another embodiment, the fusion protein is made using a single chain variable fragment (scFv), diabody, Fab, or any antibody fragment as a base scaffold to which one or two monomers of IL-10, one or two monomers of an IL-10 variant molecule, IL-2, IL-7, IL-15, IL-26, IL-27, IL-28, IL-29, IFN-alpha, TGF-beta, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or a combination thereof, is conjugated.

[0096] In one particularly preferred embodiment, the fusion protein comprises at least one variable region having a variable heavy chain (VH) and / or variable light chain (VL) linked to an IL-10 or IL-10 variant molecule. In this configuration, the fusion protein comprises an IL-10 monomer or variant IL-10 monomer linked to at least one variable region of an antibody. In one aspect, the fusion protein is a linear contiguous sequence comprising an IL-10 monomer or IL-10 monomer variant molecule linked to a VH, which is linked to a VL, which is linked to an IL-10 monomer. The antibody variable region can be a heavy chain variable (VH) region, a light chain variable (VL) region, or both. The first fusion protein comprises a protein sequence having a linear contiguous configuration in which an IL-10 monomer or variant IL-10 monomer is conjugated to the carboxy terminus of the variable region (VH or VL, or both). The second fusion protein may comprise a protein sequence having a linear, contiguous configuration in which the IL-10 monomer or variant IL-10 monomer is linked to the amino terminus of the variable region (VH or VL, or both). Representative examples of the above-mentioned first fusion protein include the following configurations: a) NH2 (Ab1VL) COOH -(linker)- NH2 (monoIL10) COOH

[0097] Representative examples of the second fusion protein include the following: NH2 (monoIL10) COOH -(linker)- NH2 (Ab1VH) COOH

[0098] When combined, the first(a) and second(b) fusion proteins form a functional protein complex in an antiparallel manner, whereby the terminally linked IL-10 or variant IL-10 monomers form a functional homodimer, and the variable regions together can form a functional antigen-binding site ("ABS") (see, e.g., Figures 9(a)-(f)).

[0099] In alternative embodiments, an IL-10 monomer or variant IL-10 monomer may be conjugated to at least two variable regions from the same antibody or two different antibodies. In this configuration, the at least two variable regions are a VH and a VL. An example of such a configuration would be a first fusion protein having a linear, contiguous protein sequence consisting of a VH region of a first antibody linked at its carboxy terminus to the amino terminus of a VL region of a second antibody, which is in turn linked to the amino terminus of an IL-10 monomer or an IL-10 variant molecule monomer. An alternative configuration would be a second fusion protein having a linear, contiguous protein sequence consisting of a monomeric IL-10 or IL-10 variant molecule monomer linked at its carboxy terminus to the amino terminus of a VH region of a second antibody, which is in turn linked to the amino terminus of a VL region of the first antibody. Representative examples of the above-mentioned first fusion proteins include the following configurations: a) NH2 (Ab 1- VH) COOH- -(linker)- NH2 (Ab2VL) COOH -(linker)- NH2 (monoIL10) COOH

[0100] Representative examples of the second fusion protein include the following: NH2 (monoIL10) COOH -(linker)- NH2 (Ab2VH) COOH -(linker)- NH2 (Ab 1- VL) COOH

[0101] When combined, the first(a) and second(b) fusion proteins form a functional protein complex in an antiparallel manner, whereby the terminally linked IL-10 or variant IL-10 monomers form a functional homodimer, and the variable regions together can form an ABS (see, e.g., Figures 9(a)-(c)).

[0102] In yet another embodiment, the fusion protein comprises two monomers of IL-10 or two monomers of variant IL-10 fused together and one or more VH and VL regions. Each monomer is individually linked to one or more VH and / or VL regions of an antibody. When more than one VH and / or VL region is used in this fusion protein configuration, the VH and VL regions may be derived from the same antibody or from at least two different antibodies. In one particular configuration of this fusion protein, the VH or VL region is linked to the amino terminus of the first monomer, which is then linked by its carboxy terminus to the amino terminus of the second monomer, which is then linked to the amino terminus of the VL or VH. Optionally, additional VH or VL regions may be linked to the amino or carboxy terminus, where the VH or VL regions may be derived from the same or different antibodies. Representative examples of the above-mentioned fusion proteins include the following structures (see, for example, Figures 10(d) to (f)). NH2 (Ab 1- VH) COOH- -(linker)- NH2 (monoIL10)COOH -(linker)- NH2 (monoIL10) COOH- (Linker) -NH2 (Ab 1- VL) COOH

[0103] The above-described fusion protein will be able to fold in a manner that allows the IL-10 monomers to form homodimers and the antibody variable domains (VH and VL) to form functional ABS.

[0104] In another embodiment, the fusion protein comprises two IL-10 or variant IL-10 monomers located at opposite ends of the fusion protein and at least one VH and VL domain, wherein the VH and VL domains are linked together. In this configuration, the VH and VL domains are fused together, and each monomer is individually linked to either the VL or VH domain of the first antibody. In this configuration, the IL-10 or variant IL-10 monomer is individually linked to either the VH or VL domain of the first antibody, respectively. Representative examples of the above-mentioned fusion proteins include the following configurations (see, for example, Figures 10(a) to 10(c)). a) NH2 (monoIL10) COOH -(linker) -NH2 (Ab 1- VH) COOH- (Linker)- NH2 (Ab 1- VL) COOH- (Linker) -NH2 (monoIL10) COOH b) NH2 (monoIL10) COOH -(linker) -NH2 (Ab 1- VL) COOH- (Linker)- NH2 (Ab 1- VH) COOH- (Linker) -NH2 (monoIL10) COOH

[0105] The IL-10 monomer or variant IL-10 monomer can be linked to the VH or VL sequence through a linker sequence. The linker can be a carboxy-terminal linker that links the carboxy-terminus of the variable chain region (VH or VL) to the amino-terminus of the IL-10 monomer or monomeric IL-10 variant molecule. Alternatively, the linker can be an amino-terminal linker that links the carboxy-terminus of the monomeric IL-10 or monomeric IL-10 variant molecule to the amino-terminus of the variable chain region (VH or VL).

[0106] Thus, in one form, the fusion protein comprises a monomeric IL-10 molecule or a variant IL-10 molecule linked to two variable regions from at least two different antibodies, where the two variable regions are configured as a VH region from a first antibody linked to a VL region from a second antibody, or a VL region from a first antibody linked to a VH region from a second antibody. Fusion proteins of this form may comprise a monomeric IL-10 molecule or a variant thereof containing at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor. The amino acid substitution affecting IL-10 receptor binding may occur in human, CMV, or EBV IL-10. The amino acid substitution may preferably be in EBV IL-10 and may include an amino acid substitution at position 31, 75, or both. The amino acid substitution may include any one or more of a V31L or A75I substitution, or both. In addition to amino acid substitutions that affect IL-10 receptor affinity, IL-10 variants may also contain modifications that affect the interdomain angle. In another embodiment, the fusion protein comprises a configuration selected from (a) a VH region of a first antibody linked at its carboxy terminus to the amino terminus of a VL region of a second antibody, followed by the carboxy terminus of an IL-10 or variant thereof monomer, or (b) an IL-10 molecule or variant thereof linked at its carboxy terminus to the amino terminus of a VH region of a second antibody, followed by the amino terminus of a VL region of the first antibody. These fusion protein configurations, in one preferred embodiment, comprise the sequences of SEQ ID NOS: 24-28, 29, and 33-53. These fusion protein sequences can form complexes, in which IL-10 monomers or variant IL-10 monomers can form homodimers. Such complexes can include and / or be configured as diabody complexes.

[0107] In another form, the fusion protein may be formed as an immunoconjugate comprising a first fusion protein comprising, at its amino terminus, the heavy chain variable region (VH) of a first antibody linked to the light chain variable region (VL) of a second antibody, which is further linked to a monomer of IL-10, and a second fusion protein comprising, at its amino terminus, a monomer of IL-10 linked to the VH of the second antibody, which is further linked to the VL of the first antibody, wherein the VH and VL of the first and second antibodies associate as a diabody and the IL-10 monomers form a functional dimeric IL-10 molecule. In another preferred embodiment, the immunoconjugate complex comprises a first fusion protein comprising at its amino terminus a VH region of a first antibody and a monomeric IL-10 molecule linked by its amino terminus, and a second fusion protein comprising at its amino terminus an IL-10 monomer linked to the VL region of the first antibody, wherein the VH region of the first antibody associates with the VL region of the first antibody, thereby enabling the monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer. The IL-10 monomer or variant IL-10 monomer may contain amino acid modifications that affect IL-10 receptor binding and / or interdomain angle, as described above. In another preferred embodiment, the immunoconjugate complex comprises a first fusion protein comprising, at its amino terminus, a VH region of a first antibody linked to a monomeric IL-10 molecule, and a second fusion protein comprising, at its amino terminus, an IL-10 monomer linked to the VL region of the first antibody, wherein the VH region of the first antibody associates with the VL region of the first antibody, thereby enabling the monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer.In yet another embodiment, the immunoconjugate comprises, at its amino terminus, a first IL-10 (or IL-10 variant molecule) monomer linked to the VH region of a first antibody, which is linked to the VL region of the first antibody, which is linked to a second IL-10 (or IL-10 variant molecule) monomer, where the two IL-10 monomers can associate together to form a functional dimer of IL-10. The recited VH and VL regions can form an antigen-binding site that specifically targets an antigen (e.g., a receptor, protein, nucleic acid, etc.). Thus, there are two chains, chain 1 and chain 2, that together form a fusion protein complex that forms a functional IL-10 (or IL-10 variant molecule) homodimer. Exemplary fusion protein chains (i.e., chain 1 and chain 2) include the following: [Table 1]

[0108] The fusion protein comprises a VH and VL pair derived from at least one antibody. The VH and VL pair serves as a scaffold to which IL-10 or its variant monomers can bind to homodimerize into a functional IL-10 molecule. Therefore, those skilled in the art will understand that the VH and VL scaffold used in the fusion protein can be selected based on the desired physical attributes required for proper IL-10 or IL-10 variant protein dimerization and / or the requirement to maintain the targeting ability of the VH and VL. Similarly, those skilled in the art will understand that the CDR regions within the VH and VL pair can also be replaced with other CDR regions to obtain a specifically targeted fusion protein. If the fusion protein is not intended to target any particular antigen, it is also envisioned that the VH and VL pair can be selected as a scaffold that does not target any particular antigen (or is an antigen with low in vivo abundance), such as a VH and VL pair derived from an anti-HIV and / or anti-Ebola antibody. The fusion protein may contain between one and four variable regions. The variable regions may be derived from the same antibody or from at least two different antibodies. The antibody variable chains may be obtained or derived from multiple antibodies (e.g., those targeting proteins, cellular receptors, and / or tumor-associated antigens, etc.). In another embodiment, the variable regions are obtained from antibodies targeting antigens associated with various diseases (e.g., cancer) or antigens that are not typically or rarely found in the serum of healthy subjects, such as variable regions from antibodies targeting EGFR, PDGFR, VEGFR, Her2Neu, FGFR, GPC3, or other tumor-associated antigens, MadCam, ICAM, VCAM, or other inflammation-associated cell surface proteins, HIV, and / or Ebola. Thus, in one embodiment, the variable region is selected from antibodies, such as, for example, an anti-EGFR antibody, an anti-MadCam antibody, an anti-HIV antibody (Chan et al., J. Virol, 2018, 92(18):e006411-19), an anti-ICAM antibody, an anti-VC antibody, and the like. The variable region is obtained or derived from an AM antibody, or an anti-Ebola antibody (U.S. Application Publication No. 2018 / 0180614, incorporated by reference in its entirety, particularly the mAbs described in Tables 2, 3, and 4). In another embodiment, the variable region is obtained or derived from an antibody that can concentrate the concentration of a cytokine, e.g., IL-10, to a particular target region, allowing IL-10 to elicit its biological effect. Such antibodies can include those that target receptors or antigens that are overexpressed or upregulated in a particular diseased region, or that are specifically expressed in a particular affected region. For example, the variable regions can be obtained from antibodies specific for various immune checkpoint targets, such as, but not limited to, epidermal growth factor receptor (EGFR), CD52, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4, CD20, CD47, GD-2, HER2, EpCAM, ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα, 5T4, Trop2, EDB-FN, TGFβ Trap, MadCam, β7 integrin subunit, α4β7 integrin, α4 ​​integrin, SR-A1, SR-A3, SR-A4, SR-A5, SR-A6, SR-B, dSR-C1, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-I1, and SR-J1, to name a few. Monomers of IL-10 (e.g., human, CMV, or EBV) or variant IL-10 molecules (as described herein) are conjugated to either the amino or carboxy terminus of the variable region (VH or VL) such that the IL-10 or variant IL-10 molecules can dimerize with each other.

[0109] The fusion protein or fusion protein complex may also have antigen targeting functionality. The fusion protein or fusion protein complex will contain VH and VL regions that can associate together to form an antigen-binding site or ABS. In some configurations, an IL-10 or IL-10 variant molecule or a monomer thereof will be covalently linked to the end containing the antigen-binding site. These targeting fusion proteins may contain at least one functional variable region or paired VH and VL at one end of the fusion protein so that the fusion protein retains the ability to target antigen and has a functional homodimer of IL-10 or IL-10 variant molecule (see Figures 9(a)-(f) and 10(a)-(f)). The variable region may be further modified by changing one or more amino acids (e.g., by addition, deletion, or substitution) to reduce antigenicity in a target. The VH and VL pair forms a scaffold onto which the CDR regions obtained from multiple antibodies can be grafted. Such antibody CDR regions include known and above-mentioned antibodies. For example, the CDR regions from any antibody can be grafted into a fusion protein that can form a VH and VL pair such as those described in SEQ ID NO: 37, 44, or 45, or a fusion protein complex such as those described in SEQ ID NO: 46 and 47, 48 and 49, or 50 and 51. The CDR regions in the above-mentioned VH and VL scaffolds include the following amino acid position numbers available for CDR grafting / insertion: [Table 2]

[0110] In another embodiment, the fusion protein described above can be represented by one of the following general formulas: 1) IL10-L 1 -X 1 -L 1 -X 2 -L 1 -IL10 (formula I), 2)(Z)n -X 1 -L 2 -Y 2 -L 1 -IL10 (formula II), 3) IL10-L 1 -Y 1 -L 2 -X 2 -(Z) n (Formula III), 4) X 1 -L 2 -X 2 -L 1 -IL10 (Formula IV), 5) IL10-L 1 -X 1 -L 2 -X 2 (Formula V), 6) X 1 -L 1 -IL10 (Formula VI), and 7) IL10-L 1 -X 2 (Formula VII) During the ceremony, "IL-10" is human IL-10 (SEQ ID NO: 1), EBV IL-10 (SEQ ID NO: 3), DV05 (SEQ ID NO: 14, 18, or 55), DV06 (SEQ ID NO: 15, 19, or 57), or DV07 (SEQ ID NO: 16, 20, or 59), and in preferred embodiments, "IL-10" consists of DV05, DV06, or DV07, more preferably, "IL-10" consists of SEQ ID NO: 55, 57, or 59, "L 1 " is a linker of SEQ ID NO: 31 or 54, "L 2 " is the linker of SEQ ID NO: 30, "X 1" is a VH region obtained from a first antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as, but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin; SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola, "X 2 " is a VL region obtained from the same antibody as X1, "Y 1 " is a VH region obtained from a second antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as, but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin; SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola, "Y 2 " is a VL region obtained from the same antibody as Y1, X and Y are derived from the same or different antibodies, "Z" is a cytokine selected from IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL_15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferon-α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13; "n" is an integer selected from 0 to 2.

[0111] In certain embodiments, the substituents of Formulas I-VII above are preferably selected from the following: IL-10 preferably consists of DV05, DV06, or DV07; more preferably, IL-10 consists of DV05, DV06, or DV07; or most preferably, IL-10 consists of SEQ ID NO: 55, 57, or 59; X1 and X2 are preferably anti-EGFR, anti-PDGFR, anti-FGFR, anti-VEGF, anti-Her2Neu, anti-GPC3, anti-MAdCAM, anti-ICAM-1, -2, -3, -4, anti-VCAM, anti-HIV, or anti-Ebola; Y1 and Y2 are preferably anti-EGFR, anti-MAdCAM, anti-ICAM-1, -2, -3, -4, anti-VCAM, anti-HIV, or anti-Ebola; Z is selected from IL-2, IL-7, or IL-15; and n is 1. In a most preferred embodiment, the fusion protein is any of SEQ ID NOs: 33-53, 61, 63, 65, or 67. Those skilled in the art will understand that the presence of the histidine tag is used in the purification process of the fusion protein and can be left intact or removed from the final product. Those skilled in the art will also understand that the VH and VL framework regions of any of the above-mentioned antibodies can be replaced with other complementarity-determining region (CDR) regions. For example, if the VH and VL regions are derived from an anti-Ebola antibody, the six CDR regions (i.e., CDRs 1-3 of both VH and VL) can be replaced with the six CDR regions of an anti-EGFR antibody (e.g., cetuximab). Thus, in one preferred embodiment, the fusion protein is SEQ ID NO: 33-34, 52, or 53. In another preferred embodiment, the fusion protein has a scaffolding represented by SEQ ID NO: 37, 44, 45, 46-47, 48-49, or 50-51, into which any six CDR regions from any antibody may be grafted. In another preferred embodiment, CDR regions from the VH and VL regions of an anti-Ebola antibody may be grafted together with CDR regions from an anti-MAdCAM, anti-VCAM, or anti-ICAM-1, -2, -3, or -4 antibody, wherein in a preferred embodiment, the CDR regions may be grafted into the fusion protein of SEQ ID NO: 37.The fusion proteins described above in Formulas II and III, IV and V, and VI and VII are designed to associate together to form biologically active homodimers of IL-10 (or variants thereof). The fusion proteins described above are designed to be either non-targeting or targeting, depending on the pair of VH and VL domains selected and / or the CDR regions grafted onto the VH and VL. The term "non-targeting" is meant to describe VH and VL domains that are unable to target a specific antigen located in vivo because the antigen is absent or the antigen-binding site (ABS) is disabled or modified to eliminate ABS functionality.

[0112] The above-mentioned fusion proteins may further be conjugated to auxiliary proteins / molecules. An auxiliary protein, as used herein, refers to a protein conjugated to a fusion protein or fusion protein complex so as to be linked to the opposite side of the IL-10 monomer or variant IL-10 monomer molecule. The addition of an auxiliary protein effectively creates a multifunctional molecule that incorporates both the functionality of the IL-10 or IL-10 variant molecule and the functionality of the auxiliary protein. The binding of an auxiliary protein (e.g., cytokines IL-2, IL-7, IL-12, IL-15, etc.) can be conjugated to a fusion protein comprising a VH and VL scaffold, for example, at the N-terminus of the VH region. For example, when applied to a fusion protein or fusion protein complex for treating a tumor, the auxiliary protein may include, but is not limited to, IL-10, an IL-10 variant molecule, IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferon-α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or any combination thereof. When applied to a fusion protein or fusion protein complex for treating an inflammatory disease, the auxiliary protein may include, but is not limited to, TGFβ. When applied to a fusion protein or fusion protein complex for treating an autoimmune disease (such as, but not limited to, fatty liver disease), the auxiliary molecule may include, but is not limited to, obticholic acid (obticholic Examples of auxiliary proteins include, but are not limited to, cyclohexyl 1,2-dihydroxybenzoates (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C36, C37, C48, C49, C51, C52, C53, C64, C75, C76, C77, C78, ​​C79, C81, C82, C83, C84, C85, C96, C97, C98, C99, C99, C99, C100, C111, C121, C131, C141, C142, C153, C154, C162, C173, C174, C175, C186, C187, C188, C189, C196, C197, C198, C199, C211, C222, C233, C244, C255, C256, C257, C264, C265, C276, C277, C280, C281, C298, C299, C378, C379, C481, C499, C51, C52, C64, C77, C78, ​​C79, C81, C99, C99, C99, C199, C199, C211, C222, C233, C244, C257, C265, C277, C278, C281, C299, C37

[0113] The fusion proteins described above can also contain additional amino acid sequences that aid in the recovery or purification of the fusion protein during the manufacturing process. These may include various sequence modifications or affinity tags, such as, but not limited to, Protein A, albumin-binding protein, alkaline phosphatase, FLAG epitope, galactose-binding protein, histidine tag, and any other tag known in the art. See, for example, Kimple et al. (Curr. Protoc. Protein Sci., 2013, 73:Unit 9.9, Table 9.91, incorporated by reference in its entirety). In one aspect, the affinity tag is a histidine tag having the amino acid sequence HHHHHH (SEQ ID NO: 32). The histidine tag may be removed from the final product or left intact. In another embodiment, the affinity tag is a Protein A modification incorporated into the fusion protein (e.g., the VH region of a fusion protein described herein), such as those set forth in SEQ ID NOs: 34 or 44-53. Those skilled in the art will appreciate that any of the fusion protein sequences described herein can be modified to incorporate Protein A modifications by inserting point amino acid substitutions within antibody framework regions as described in the art.

[0114] In yet another embodiment, the various fusion proteins described above can be used in methods of treating cancer, treating or preventing IBD or Crohn's disease, autoimmune diseases, NAFLD, or NASH. IL-10 variant polynucleotides

[0115] Polynucleotide sequences encoding the above-described variant IL-10 molecules and various fusion proteins and / or immunocytokines are also included within the scope of the present application. Upon localizing the region for the key IL-10 receptor-binding region and / or interdomain angle in the variant IL-10 molecules of the present application, the DNA modifications necessary to implement the desired modifications to the amino acid sequence are within the skill of one of ordinary skill in the art. Such modifications would be made using conventional recombinant DNA techniques and methods. For example, specific amino acid sequence additions or substitutions can be introduced into the IL-10 sequence at the nucleic acid (DNA) level using site-directed mutagenesis methods using synthetic oligonucleotides, methods that are also well known in the art.

[0116] In another embodiment, polynucleotides encoding variant IL-10 sequences can be produced using standard techniques in molecular biology. For example, polynucleotide sequences encoding the above-described variant molecules can be obtained using recombinant methods, for example, by screening cDNA and genomic libraries obtained from cells expressing the gene, or by deriving the gene from a vector known to contain it. The gene of interest can also be produced synthetically rather than by cloning, based on a known sequence. Molecules can be designed with appropriate codons for a particular sequence. The complete sequence is then assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et al., J. Biol. Chem. (1984) 259:6311. sea ​​bream.

[0117] In one embodiment, the IL-10 variant molecule or fusion protein thereof is a nucleic acid molecule encoding any of SEQ ID NOs: 9-29, 33-53, 55, 57, or 59. In another embodiment, the IL-10 variant molecule is DV05, DV06, or DV07 of SEQ ID NOs: 56, 58, or 60, respectively. Nucleic acid molecules encoding DV05, DV06, or DV07 can contain insertions, deletions, or substitutions (e.g., degenerate codons) that do not alter the functionality of the IL-10 variant molecule. Nucleotide sequences encoding the IL-10 variants and fusion proteins described herein may differ from the sequences of SEQ ID NOs: 1, 3, 5, 7, 9-29, 33-53, 55, 57, 59, 61, 63, 65, or 67 due to the degeneracy of the genetic code, and may be 70-99%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the foregoing sequences. Nucleotide sequences encoding the IL-10 variants and fusion proteins of SEQ ID NOs: 1, 3, 5, 7, 9-29, 33-53, 55, 57, 59, 61, 63, 65, or 67 can also be 70% to 99%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous due to the insertion, deletion, or substitution of at least one nucleotide. Nucleotide sequences having 70% to 99%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 56, 58, 60, 62, 64, 66, and 68 due to the insertion, addition, deletion, or substitution of at least one nucleotide are also contemplated in the present application. Additionally, the nucleotide sequences encoding the IL-10 variants and fusion proteins described herein may further comprise well-known sequences that aid in, for example, protein expression, production, or secretion. Such sequences may include, for example, a leader sequence, a signal peptide, and / or a translation initiation site / sequence (e.g., a Kozak consensus sequence). The nucleotide sequences described herein may also include one of a number of restriction enzyme sites to allow insertion into various expression systems / vectors.

[0118] In another embodiment, the polynucleotide is carried in a vector containing the desired IL-10 sequence or is artificially synthesized using oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques. See, e.g., Sambrook (supra). In another embodiment, the nucleotide sequence encoding the variant IL-10 molecule is obtained through a process of annealing complementary overlapping synthetic oligonucleotides produced in an automated polynucleotide synthesizer, followed by ligation and amplification of the ligated nucleotide sequence by PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. In addition, oligonucleotides can be synthesized by PCR. Nucleotide-directed synthesis (Jones et al., Nature (1986) 54:75-82), existing nucleoside Oligonucleotide-directed mutagenesis of the nucleotide region (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic filling of gapped oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033). can be used to obtain molecules for use in the subject methods.

[0119] A variety of suitable expression vectors are available, which are well known to those skilled in the art, and can be used to express and introduce variant IL-10 molecules and fusion proteins. Examples of such vectors include pUC-type vectors, pBR-type vectors, pBI-type vectors, pGA-type vectors, pBinl9, pBI121, pGreen series, pCAMBRIA series, pPZP series, pPCV001, pGA482, pCLD04541, pBIBAC series, pYLTAC series, pSB11, pSB1, pGPTV series, and viral vectors.

[0120] A vector carrying an IL-10 variant molecule may also contain other vector components required for vector functionality. For example, the vector may contain a signal sequence, a tag sequence, a protease-specific sequence, a selection marker, and other regulatory sequences, such as a promoter, required for proper replication and expression of the variant IL-10 molecule. The specific promoter used in the vector is not particularly limited, as long as it can induce expression of the variant IL-10 molecule in various host cell types. Similarly, the type of tag promoter is not particularly limited, as long as the tag sequence serves to facilitate simpler or easier purification of the expressed variant IL-10 molecule. Examples of such promoters include hexa-histidine, GST, MBP, HAT, HN, S, TF, Trx, Nus, biotin, FLAG, myc, RCFP, GFP, and the like. The protease recognition sequence is not particularly limited, and recognition sequences for factor Xa, thrombin, HRV, 3C protease, and the like can be used. The marker to be selected is not particularly limited as long as it allows the detection of transformed rice cells, and for example, a neomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, etc. can be used.

[0121] The resulting DNA construct carrying the desired IL-10 variant or fusion protein can then be used directly in gene therapy or to produce a recombinant IL-10 variant or fusion protein. In one embodiment, the variant IL-10 molecule or fusion protein of the present application can be delivered by any method known in the art, including direct administration of the mutant IL-10 protein and gene therapy using a vector encoding the mutant IL-10 protein. Gene therapy can be achieved using plasmid DNA or viral vectors, such as adeno-associated viral vectors, adenoviral vectors, retroviral vectors, etc. In some embodiments, the viral vectors of the present application are administered as viral particles, while in other embodiments, they are administered as plasmids (e.g., as "naked" DNA).

[0122] Other methods for delivery of nucleotide sequences include those known in the art. These may include delivery of nucleotide sequences, such as, but not limited to, DNA, RNA, siRNA, mRNA, oligonucleotides, or variants thereof, encoding IL-10 or IL-10 variant molecules via cell-penetrating peptides, hydrophobic moieties, electrostatic complexes, liposomes, ligands, liposomal nanoparticles, lipoproteins (preferably HDL or LDL), folate-targeted liposomes, antibodies (e.g., folate receptors, transferrin receptors), targeting peptides, or via aptamers. Nucleotide sequences encoding IL-10 variant molecules may be delivered to a subject via direct injection, infusion, patch, bandage, mist, or aerosol, or via thin film delivery. The nucleotide (or protein) may be directed to any area where targeted delivery of cytokine stimulation is desired. These may include, for example, the lungs, gastrointestinal tract, skin, liver, brain via intracranial injection, and deep-seated metastatic tumor sites via ultrasound-guided injection. Testing for IL-10 variants

[0123] In one embodiment, variant IL-10 molecules or fusion proteins thereof will be screened for novel functions not previously generated using the IL-10 homodimer sequence, such as suppressing the secretion of inflammatory cytokines by macrophages but not activating T cells. In one preferred embodiment, variant IL-10 molecules or fusion proteins thereof will be based on the EBV-IL10 backbone, which has an anti-inflammatory response but lacks the ability to stimulate T cells. These variant EBV-IL10 molecules or fusion proteins thereof will contain modifications to the receptor-binding domain and the linking region that alter the primary, secondary, and tertiary structure, previously unexplored, to restrict or widen the angle between IL-10 homodimers. In another embodiment, IL-10 variant molecules or fusion proteins thereof containing modifications to the linking region or the region involved in forming the interdomain angle will have enhanced CD8+ T cell function. In another embodiment, IL-10 variant molecules or fusion proteins thereof containing modifications to the linking region or the region involved in forming the interdomain angle will have suppressed myeloid function but enhanced Kupffer cell function.

[0124] After a variant IL-10 molecule or fusion protein thereof has been constructed and expressed, one skilled in the art can perform screening assays on the IL-10 variant molecule or fusion protein thereof to determine whether the molecule has the desired biological function conferred by the modification to the IL-10 receptor-binding region and / or interdomain angle. Multiple screening assays for testing for the desired biological function are known and available to those skilled in the art. In one embodiment, the desired biological function includes, but is not limited to, reduced anti-inflammatory response, reduced T cell stimulation, enhanced T cell function, enhanced Kupffer cell functionality, and reduced mast cell degranulation.

[0125] For example, exposure to IL-10 is known to stimulate T cells to produce and secrete more IFNγ upon T cell receptor stimulation. At the same time, IL-10 exposure prevents the secretion of TNFα, IL-6, and other pro-inflammatory cytokines secreted by monocytes / macrophages in response to LPS. IL-10 also inhibits FoxP3. + CD4 + T reg In one embodiment, an IL-10 variant molecule or fusion protein thereof that maximizes monocyte / macrophage suppression but lacks T cell effects, including both stimulatory and inhibitory responses, would be positively selected. In one embodiment, screening for an IL-10 variant molecule or fusion protein thereof with increased anti-inflammatory effects would be positively selected for the treatment of autoimmune disease, anti-inflammatory disease, or both. In another embodiment, screening for an IL-10 variant molecule or fusion protein thereof that maximizes Kupffer cell elimination and suppresses T reg IL-10 variant molecules or fusion proteins thereof lacking suppression would also be selected for development for the treatment of nonalcoholic steatohepatitis (NASH) and / or nonalcoholic fatty liver disease (NAFLD). In yet another embodiment, IL-10 variants that maximize T cell biology, including both stimulatory and suppressive responses, and also have enhanced Kupffer cell elimination would be selected for development for the treatment of cancer.

[0126] The literature has shown that cells of the immune system, e.g., T cells, monocytes / macrophages, Kupffer cells, T reg The literature is replete with descriptions of assaying the effects of cytokines on cells, such as mast cells, etc. The present application adapts these assay systems to test biological responses using similar assays by contacting the variant IL-10 molecules or fusion proteins thereof of the present application.

[0127] Various methods for assaying the effectiveness of eliciting a T cell response have been described in the prior art. Any one of these methods can be applied to testing the variant IL-10 molecules described herein. For example, Chan et al. (2015) describes one such method that can be applied to variant IL-10 molecules: CD8 + T cells are isolated from peripheral blood mononuclear cells (PBMCs) using anti-CD8 microbeads. + T cells are activated using anti-CD3 and anti-CD28 antibodies. For example, activation can occur using plates coated with at least about 5-20 micrograms / mL of anti-CD3 antibody and at least about 1-5 micrograms / mL of anti-CD28 antibody for a period of about 3 days. Following activation, T cells are harvested, plated, and treated with an EBV-IL10 variant or its fusion protein for a period of about 3-5 days. Commercially available PEGylated recombinant human IL-10 or EBV-IL10 can be used as a control. Following treatment with the EBV-IL10 variant, T cells are treated with soluble anti-CD3. Following treatment with anti-CD3, cell culture medium is collected and assayed for interferon gamma (IFNγ) secretion by ELISA.

[0128] Various methods for assaying monocyte / macrophage stimulation by cytokines have been described in the prior art. Any one of these methods can be applied to testing the variant IL-10 molecules described herein. For example, Conway et al. (2017) describes one such method that can be applied to variant IL-10 molecules. described a method such as this. Human monocytes were isolated from the buffy coat of fresh donor blood using a Ficoll gradient, followed by hyperosmolar density centrifugation in Percoll. After 30 minutes of culture in RPMI supplemented with 5% human serum and 1% L-glutamine, the monocytes became adherent and were washed with SMEM spinner medium to remove contaminating lymphocytes. Solutions and materials were tested to confirm the absence of LPS. After 4 days of culture, monocytes / macrophages were contacted with 10 ng / ml LPS and different concentrations of variant IL-10 molecules and incubated with them for at least 24 hours. Culture supernatants were collected, and TNF-α and IL-1β concentrations were determined by ELISA.

[0129] Various methods for assaying Kupffer cell responses to cytokines have been described in the prior art. Any one of these methods can be applied to test the variant IL-10 molecules described herein. For example, Chan et al. (2016) described one such method, which can be applied to variant IL-10 molecules. Kupffer cells were seeded into 24- or 96-well plates and incubated overnight in hepatocyte incubation medium (phenol red-free RPMI, pen / strep, Cell Maintenance Supplement B (Invitrogen)). The cells were washed and exposed to variant IL-10 molecules for 24 hours. The cells were washed once and exposed to 15–20 μl of DiI-LDL, DiI-VLDL, DiI-OxLDL, or DiI-AcLDL, 2 μl DMSO, and 15 μl cytochalasin D, and uptake was measured after 4 hours. All cells were washed once with 1x PBS and lysed in 110 μl of cell lysis buffer. 45 μl of the cell lysate was transferred to a clear-bottom, black-walled plate, and fluorescence was read at 575 nm.

[0130] Various methods for testing the effectiveness of using cytokines to stimulate regulatory T cell responses have been described in the prior art.Any one of these methods can be applied to test the variant IL-10 molecules described herein.For example, Chan et al. (2016) describes one such method that can be applied to variant IL-10 molecules.CD4 + T cells, CD4 + Cells were isolated with microbeads and cultured for 5-6 days in AIMV medium containing various concentrations of variant IL-10 molecules, as well as 2 micrograms / mL immobilized anti-CD3 and 1 mg / mL anti-CD28. Cells were analyzed for FoxP3 expression by flow cytometry analysis, demonstrating that TGF-β or IL-2 induce the upregulation of FoxP3. + CD4 + Determine whether it is induced in regulatory T cells.

[0131] Various methods for assaying the effectiveness of mast cell proliferation in response to cytokine stimulation have been described in the prior art. The murine mast cell line MC / 9 is a common cell line used to generate IL-10 molecule release tests. Specifically, IL-10 and IL-10 variant molecules induce dose-titrative proliferation of mast cells. In contrast, IL-10 inhibits Fc expression by mast cells, suggesting that IL-10 exerts both stimulatory and inhibitory effects on these cells. Any one of these methods can be applied to test the variant IL-10 molecules described herein. For example, Thompson-Snipes et al. (1991) reported: One such method, which can be applied to variant IL-10 molecules, is described. MC / 9 mast cells are seeded into flat-bottom 24-well plates containing 1 ml of RPMI 1640, 10% FCS, 50 mM 2-ME, and various concentrations of variant cytokines. After 3 days of culture, cells are counted using a cell counter to determine the effect of the variant IL-10 molecules on mast cell proliferation.

[0132] IL-10 is known to play a role in the expression of IgE receptors, FcεRI, and the inhibition of IgE-mediated cytokine production by mast cells. Therefore, methods for testing the effects of IL-10 on mast cells have been described in the prior art. These methods can be applied to testing the IL-10 variant molecules described herein. For example, Kennedy Norton et al. (2008) reported that one described such a method. Human mast cells were isolated from donor skin samples and cultured in medium containing stem cell factor (SCF) in the presence or absence of IL-10. FcεRI expression was determined by flow cytometry using an anti-FcεRI-specific antibody followed by FITC-labeled anti-mouse F(ab')2. Compositions and formulations comprising IL-10 variant molecules

[0133] The IL-10 variant molecule or fusion protein of the present application can also be formulated in a pharmaceutical composition comprising a therapeutically effective amount of the variant IL-10 molecule and a pharmaceutical carrier and / or pharmaceutically acceptable excipient. The pharmaceutical composition can be formulated using commonly used buffers, excipients, preservatives, and stabilizers. The pharmaceutical composition is formulated for administration to a patient in a therapeutically effective amount sufficient to provide the desired therapeutic result. Preferably, such an amount has minimal negative side effects. In one embodiment, the amount of the administered variant IL-10 molecule or fusion protein thereof is sufficient to treat an inflammatory disease or condition. In another embodiment, the amount of the administered variant IL-10 molecule or fusion protein thereof is sufficient to treat cancer. The amount administered may vary from patient to patient and will need to be determined by considering the subject's or patient's disease or condition, the patient's overall health, the method of administration, the severity of side effects, etc. In a preferred embodiment, the pharmaceutical composition will comprise a variant IL-10 molecule or fusion protein thereof containing one or both modifications to the receptor-binding domain and / or the interdomain angle of IL-10. In another embodiment, the variant IL-10 molecule is a PEGylated form of the variant IL-10 molecule. In an even more preferred embodiment, the pharmaceutical composition comprises a variant IL-10 molecule incorporated as a fusion protein or immunocytokine and a pharmaceutical excipient.

[0134] The effective amount for specific patient can vary according to factors such as the condition being treated, the overall health condition of patient, the method route and dosage of administration and the severity of side effects.The appropriate dosage to be administered to patient is usually determined by clinician using the parameters or factors that are known or suspected in the art to affect treatment, or that are predicted to affect treatment.Generally, dosage is started at a dose somewhat below optimal dosage, and then it is increased by small increments until it achieves the desired or optimal effect against any negative side effects.Important diagnostic measures include, for example, the symptoms of inflammation or the level of inflammatory cytokines produced.

[0135] The dose administered to a patient can also be optimized based on the addition of certain serum half-life extending modifications to the IL-10 or variant IL-10 molecule (see, e.g., pegylated IL-10, discussed in detail in U.S. Pat. Nos. 9,943,568, 10,010,588, and 10,143,726, which is known to improve the circulating half-life of IL-10). Fusion proteins, immunoconjugates, fusion proteins, minibodies, and diabodies comprising IL-10 or variant IL-10 disclosed herein can also extend circulating half-life while retaining high affinity binding to the IL-10 receptor. Thus, in one embodiment, various diseases, disorders, or conditions associated with IL-10, or that can be ameliorated by administering a fusion protein, fusion protein complex, immunoconjugate, or diabody comprising IL-10 or variant IL-10, can be administered to a patient in need thereof. In one preferred embodiment, IL-10-associated diseases, disorders, or conditions can be treated or prevented by administering to a patient in need thereof a therapeutically effective amount of an immunoconjugate complex, fusion protein, or diabody having EBV IL-10 or a variant thereof (including variants that affect IL-10 receptor binding affinity), wherein the immunoconjugate complex, fusion protein, or diabody has a molecular weight of approximately 60-155 kDa and the therapeutically effective amount is within the range of approximately 0.5 micrograms / kilogram to 100 micrograms / kilogram. The immunoconjugates, fusion proteins, or diabodies described herein can be administered daily, three times a week, twice a week, weekly, twice a month, or monthly. The EBV IL-10 portion and variable regions of the immunoconjugate, fusion protein, or diabody can be in any configuration or combination of the structures discussed herein.The half-life extended molecules described herein will be effective in treating a variety of diseases, including, but not limited to, cancer, inflammatory diseases, autoimmune diseases (e.g., non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD)), and elevated cholesterol.

[0136] Methods for co-administration or treatment with a second therapeutic agent, such as a cytokine, steroid, chemotherapeutic agent, antibiotic, anti-inflammatory agent, or radiation, are well known in the art. These may include one or more of the following: interferon-β, e.g., IFNβ-1α and IFN-β-1β, myelin basic protein-stimulating protein, corticosteroids, IL-1 inhibitors, TNF inhibitors, anti-TNFα antibodies, anti-IL-6 antibodies, IL-1br-Ig fusions, anti-IL-23 antibodies, antibodies to CD40 ligand and CD80, antagonists of IL-12 and IL-23, e.g., antagonists of the p40 subunit of IL-12 and IL-23 (e.g., inhibitory antibodies to the p40 subunit), IL-22 antagonists, small molecule inhibitors, e.g., methotrexate, leflunomide, scylotecan, cyclosporine ... This may include combination treatment with other therapeutic agents such as, but not limited to, limus (rapamycin) and its analogs, e.g., CCI-779, Cox-2 and cPLA2 inhibitors, NSAIDs, p38 inhibitors, TPL-2, Mk-2, NFkβ inhibitors, RAGE or soluble RAGE, P-selectin or PSGL-1 inhibitors (e.g., small molecule inhibitors, antibodies thereto, e.g., antibodies to P-selectin), estrogen receptor beta (ERB) agonists, or ERB-NFkβ antagonists.

[0137] Additionally, combination treatments useful with the administration of an IL-10 variant molecule or fusion protein thereof may include TNF inhibitors, such as chimeric, humanized, effectively human, human, or in vitro-generated antibodies that bind to TNF, or antigen-binding fragments thereof, soluble fragments of TNF receptors, such as p55 or p75 human TNF receptors or derivatives thereof, such as 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL™), p55 kD TNF receptor-IgG fusion protein, and TNF enzyme antagonists, such as TNFα-converting enzyme (TACE) inhibitors. Other combination treatments with anti-inflammatory agents / drugs, including, but not limited to, standard nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclo-oxygenase-2 inhibitors. NSAIDs can include aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, and / or tolmetin. The cyclo-oxygenase-2 inhibitor used in the compositions according to the present application can be, for example, celecoxib or rofecoxib.

[0138] Additional therapeutic agents that may be co-administered and / or co-formulated with the IL-10 variant molecule or fusion protein thereof include interferon-β, e.g., IFN β-1α and IFN beta-1β, COPAXONE®, corticosteroids, IL-1 inhibitors, TNF antagonists (e.g., soluble fragments of TNF receptors, e.g., p55 or p75 human TNF receptors or derivatives thereof, e.g., 75kdTNFR-IgG, antibodies to CD40 ligand and CD80, and antagonists of IL-12 and / or IL-23, e.g., antagonists of the p40 subunit of IL-12 and IL-23 (e.g., inhibitory antibodies that bind to the p40 subunit of IL-12 and IL-23), methotrexate, leflunomide, and sirolimus (rapamycin), or analogs thereof, e.g., CCI-779. Other therapeutic agents can include Imfinzi or Atezolizumab.

[0139] For the purpose of treating NASH, for example, the IL-10 variant molecule or its fusion protein can be combined with cholesterol-lowering agents, such as statins and non-statin drugs.These drugs include, but are not limited to, simvastatin, atorvastatin, rosuvastatin, lovastatin, pravastatin, gemfibrozil, fluvastatin, cholestyramine, fenofibrate, cholesterol absorption inhibitors, bile acid binding resins or sequestrants, and / or microsomal triglyceride transfer protein (MTP) inhibitors.

[0140] An effective amount of a therapeutic agent will affect the level of inflammation or a disease or condition by alleviating symptoms, for example, an effect can include an effect of at least 10%, at least 20%, at least about 30%, at least 40%, at least 50%, or more, such that the disease or condition is reduced or completely treated.

[0141] The pharmaceutical composition containing the variant IL-10 molecule or its fusion protein is mixed with a pharmaceutically acceptable carrier or excipient. Various pharmaceutical carriers are known in the art and can be used in the pharmaceutical composition. For example, the carrier can be any compatible non-toxic substance suitable for delivering the variant IL-10 molecule composition of the present application to a patient. Examples of suitable carriers include saline, Ringer's solution, dextrose solution, and Hank's solution. Carriers can include any poloxamer commonly known to those skilled in the art, including, but not limited to, those having molecular weights of 2900 (L64), 3400 (P65), 4200 (P84), 4600 (P85), 11,400 (F88), 4950 (P103), 5900 (P104), 6500 (P105), 14,600 (F108), 5750 (P123), and 12,600 (F127). Carriers can also include emulsifiers, including, but not limited to, polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, to name a few. Non-aqueous carriers, such as fixed oils and ethyl oleate, can also be used. Carriers can also include additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives; see, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984). Formulations of therapeutic and diagnostic agents can be prepared, e.g., by administering to a subject in need thereof a pharmaceutical composition comprising: The pharmaceutical composition can be prepared in the form of a liquid, lyophilized powder, slurry, aqueous solution, or suspension by mixing with a physiologically acceptable carrier, excipient, or stabilizer.

[0142] The compositions of the present application can be administered orally or injected into the body. Formulations for oral use may also include compounds to further protect the variant IL-10 molecule from proteases in the digestive tract. Injections are usually intramuscular, subcutaneous, intradermal, or intravenous. Alternatively, intraarticular injection or other routes can be used in appropriate circumstances. Variant IL-10 molecules administered parenterally are preferably formulated in a unit-dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutical carrier and / or a pharmaceutically acceptable excipient. In other embodiments, the compositions of the present application can be introduced into the patient's body by an implantable or injectable drug delivery system. Therapeutic Uses of IL-10 Variants

[0143] In one embodiment, the present application provides a method for treating, alleviating, or reducing symptoms associated with inflammation, an inflammatory disease, or an autoimmune disease. The present application also provides IL-10, an IL-10 variant molecule, a fusion protein thereof, or a chimeric molecule thereof for use as a pharmaceutical for inflammation, an inflammatory disease, an autoimmune disease, cancer, or a tumor. The present application also contemplates the use of IL-10, an IL-10 variant molecule, a fusion protein thereof, or a chimeric molecule thereof for use in treating inflammation, an inflammatory disease, an autoimmune disease, cancer, or a tumor, including, for example, IBD, Crohn's disease, ulcerative colitis, NASH, NAFLD, hypercholesterolemia, or cancer, to name a few. The method contemplates administering a therapeutically effective amount of one or more of the variant IL-10 molecules or fusion proteins thereof described herein. In one embodiment, the present application includes a method of treating an inflammatory or autoimmune disease, comprising administering a therapeutically effective amount of a variant IL-10 molecule comprising one or more modifications associated with the receptor-binding domain and / or a region involved in forming the interdomain angle. In a preferred embodiment, the method comprises administering a variant EBV-IL10 molecule or a fusion protein thereof. In a preferred embodiment, a variant IL-10 molecule or a fusion protein thereof useful for treating an inflammatory disease comprises a variant molecule having a restricted interdomain angle and / or exhibiting reduced receptor affinity compared to the wild-type IL-10 molecule. In other embodiments, a variant IL-10 molecule or a fusion protein thereof useful for treating an inflammatory disease comprises a variant molecule having a relaxed interdomain angle and / or exhibiting reduced receptor affinity compared to the wild-type IL-10 molecule. PEGylated forms of variant IL-10 molecules are also contemplated as part of the present application for inflammatory diseases or inflammation.

[0144] Inflammatory or autoimmune diseases of the present application include any disease or condition associated with unwanted or undesired inflammation and immune responses, including, but not limited to, inflammatory bowel disease (IBD), Crohn's disease, psoriasis, rheumatoid arthritis, nonalcoholic fatty liver disease (NAFLD), or nonalcoholic steatohepatitis (NASH). In other embodiments, the disease or condition includes a neurodegenerative disorder, e.g., Parkinson's disease, amyelotrophic lateral sclerosis (ALS), fatal familial insomnia, Rasmussen's encephalitis, Down's syndrome, Huntington's disease, Gerstmann-Sträussler-Scheinker disease, tuberous sclerosis, neuronal ceroid lipofuscinosis, subacute sclerosing panencephalitis, Lyme disease, Tsetse disease (African sleeping sickness), HIV dementia, bovine spongiform encephalopathy ("mad cow" disease), Creutzfeldt-Jakob disease, herpes simplex encephalitis, herpes zoster cerebellitis, progressive paralysis (syphilis), tuberculous meningitis, tuberculous encephalitis, optic neuritis, granulomatous vasculitis, temporal arthritis, cerebral vasculitis, spat's syndrome, or other conditions. Spatz-Lindenberg's disease, methamphetamine-associated vasculitis, cocaine-associated vasculitis, traumatic brain injury, stroke, Lance-Adams syndrome, post-anoxic encephalopathy, radiation necrosis, limbic encephalitis, Alzheimer's disease, progressive supranuclear palsy, striatonigral degeneration, corticobasal ganglionic degeneration, primary progressive aphasia, chromosome 17-linked frontotemporal dementia, spinal muscular atrophy, HIV-associated myelopathy, HTLV-1-associated myelopathy (tropical spastic paraparesis), tabes dorsalis (syphilis), transverse myelitis, post-polio syndrome, spinal cord injury, radiation myelopathy, Charcot-Marie-Tooth disease, HIV-associated polyneuropathy, Campylobacter-associated motor axonopathy, chronic inflammatory demyelinating polyneuropathy, and diabetic amyotrophy. Detachment, phantom limb, complex regional pain syndrome, diabetic neuropathy, paraneoplastic neuropathy, myotonic dystrophy, HTLV-1-associated myopathy, trichinosis, inflammatory myopathy (polymyositis, inclusion body myositis, dermatomyositis), sickle cell disease, alpha-1-antitrypsin deficiency, tuberculosis, subacute bacterial endocarditis, chronic viral hepatitis, viral cardiomyopathy, Chagas disease, malaria, Coxsackie B infection, macular degeneration These include, but are not limited to, rheumatoid arthritis, retinitis pigmentosa, vasculitis, inflammatory bowel disease, rheumatoid arthritis, bullous pemphigus, Churg-Strauss syndrome, myocardial infarction, toxic epidermal necrolysis, shock (e.g., acute anaphylactic shock), type 1 diabetes, autoimmune thyroiditis, lymphoma, ovarian cancer, lupus (systemic lupus erythematosus), asthma, progeria, sarcoidosis, type 2 diabetes, and metabolic syndrome.Other diseases or conditions associated with inflammation that are embodiments of the application include inflammatory lung disorders such as bronchitis, oxidant-induced lung injury and chronic obstructive airway disease, inflammatory disorders of the eye including corneal dystrophies, ocular hypertension, trachoma, onchocerciasis, retinitis, uveitis, sympathetic ophthalmia, and endophthalmitis, chronic inflammatory disorders of the gums including periodontitis, chronic inflammatory disorders of the joints including arthritis, septic arthritis and osteoarthritis, tuberculous arthritis, leprous arthritis, sarcoid arthritis, disorders of the skin including sclerodermatitis, sunburn, psoriasis, and eczema, autoimmune diseases including encephalomyelitis and viral or autoimmune encephalitis, immune complex vasculitis, and diseases of the heart including ischemic heart disease, heart failure, and cardiomyopathies. Other non-limiting examples of diseases that may benefit from a variant IL-10 molecule or fusion protein thereof include adrenal insufficiency, hypercholesterolemia, atherosclerosis, bone diseases associated with increased bone resorption such as osteoporosis, pre-eclampsia, eclampsia, uremic complications, chronic liver failure, and other disorders associated with inflammation such as cystic fibrosis, tuberculosis, cachexia, ischeimia / reperfusion, hemodialysis-related conditions, glomerulonephritis, restenosis, inflammatory sequelae of viral infections, hypoxia, hyperbaric seizures and toxicity, dementia, Sydenham's chorea, Huntington's disease, epilepsy, Korsakoff's disease, intellectual disability associated with cerebrovascular accidents, NO-mediated brain damage and related sequelae, ischemic cerebral edema (stroke), migraine, vomiting, immune complex diseases, allograft rejection, infections caused by invasive microorganisms, and aging.

[0145] The most effective IL-10 variants or fusion proteins thereof for treating anti-inflammatory diseases or conditions include those with reduced ability to stimulate T cells. Accordingly, the present inventors have shown that modifying the receptor-binding domain through an amino acid substitution at position 75 induces the least amount of T cell stimulation. Specifically, EBV IL-10 with an A75I substitution in SEQ ID NO:3 (or SEQ ID NO:57) has been shown to reduce T cell stimulation (see, e.g., Figure 8E, designated DV06). Accordingly, one particularly preferred embodiment contemplates the use of diabodies and monobodies comprising IL-10 variant molecules with a DV06-based mutation (a substitution at amino acid position 75 of SEQ ID NO:3 or SEQ ID NO:57). In a more preferred embodiment, the inflammatory disease method will utilize a fusion protein or fusion protein complex comprising SEQ ID NOs:26-27, 37, 40, 41-42, 43, 48-49, or a combination thereof.

[0146] In another embodiment of the present application, a method of treatment comprises administering IL-10 or a variant IL-10 molecule, or a fusion protein thereof, to treat or reduce symptoms associated with cancer. The method contemplates administering a therapeutically effective amount of one or more of the IL-10 molecules, variant IL-10 molecules, or fusion proteins thereof described herein. In one embodiment, the present application includes a method of treating or reducing symptoms associated with cancer, comprising administering a therapeutically effective amount of a variant IL-10 molecule comprising one or more modifications associated with the receptor-binding domain and / or a region involved in forming the inter-domain angle. In a preferred embodiment, the method comprises administering a variant EBV-IL10 molecule, or a fusion protein thereof. A variant IL-10 molecule, or a fusion protein thereof, useful for treating cancer includes a variant molecule that has a restricted inter-domain angle and / or also exhibits high receptor affinity compared to the wild-type IL-10 molecule. Variant IL-10 molecules or fusion proteins thereof useful for treating or reducing symptoms associated with cancer include variant molecules that have a relaxed interdomain angle and / or exhibit high receptor affinity compared to wild-type IL-10 molecules. PEGylated forms of variant IL-10 molecules are also contemplated as part of the present application for treating cancer. One specific example of a fusion protein that can reduce tumor volume in vivo includes an IL-10 variant with two substitutions at amino acid positions 31 and 75 of SEQ ID NO: 3, including the specific substitutions V31L and A75I, designated DV07 (e.g., SEQ ID NO: 59). Figures 16A-C show that, at various doses, a fusion protein comprising a DV07 EBV IL-10 molecule conjugated to a diabody construct designated D:DV07 reduced tumor volume over a 7- and 10-day time course. Thus, one particularly preferred embodiment contemplates the use of diabodies and monobodies with IL-10 variant molecules with DV07-based mutations (substitutions at amino acid positions 31 and 75 of SEQ ID NO:3 or SEQ ID NO:59).In a more preferred embodiment, the method of treating cancer or tumor or tumour utilizes a fusion protein or fusion protein complex comprising SEQ ID NO: 28-29, 33, 34, 35-36, 38-39, 46-47, 61, 63, 65, or 67, or a combination thereof.

[0147] Cancers or proliferative disorders that can be treated by the variant IL-10 molecules or fusion proteins thereof described herein include various forms of cancer, including, but not limited to, cancer of the uterus, cervix, breast, prostate, testis, penis, gastrointestinal tract, e.g., esophagus, oropharynx, stomach, small or large intestine, colon, or rectum, kidney, renal cell, bladder, bone, bone marrow, skin, head and neck, skin, liver, gallbladder, heart, lung, pancreas, salivary gland, adrenal gland, thyroid, brain, e.g., glioma, ganglion, central nervous system (CNS) and peripheral nervous system (PNS), and immune system, e.g., spleen or thymus. The present application provides methods for treating, for example, immunogenic tumors, non-immunogenic tumors, latent tumors, virally induced cancers, e.g., epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papilloma virus, adenocarcinoma, lymphoma, carcinoma, melanoma, leukemia, myeloma, sarcoma, teratocarcinoma, chemically induced cancer, metastasis, and angiogenesis. The present application also provides methods for treating, for example, regulatory T cells (T reg ) and / or CD8 + By modulating the activity of T cells, it is intended to reduce tolerance to tumor cells or cancer cell antigens. In a preferred embodiment, the IL-10 variant molecule is particularly useful for treating patients or subjects with liver metastatic disease.

[0148] In yet other embodiments, methods of treating or reducing symptoms associated with inflammatory disease or cancer include administering a variant IL-10 molecule or a fusion protein thereof or a derivatized form thereof (e.g., PEGylated) in combination with other therapeutic agents, including, but not limited to, cytokines or cytokine antagonists such as IL-12, IL-2, IL-15, interferon-alpha, or anti-epidermal growth factor receptor, doxorubicin, epirubicin, antifolates such as methotrexate or fluorouracil, irinotecan, cyclophosphamide, radiation therapy, hormone or antihormonal therapy such as androgens, estrogens, antiestrogens, flutamide, or diethylstilbestrol, surgery, tamoxifen, ifosfamide, mitolactol, azathioprine, riboflavin, riboflavin, riboflavin, riboflavin inhibitors ... Examples of therapeutic agents include alkylating agents such as melphalan or cis-platin, etoposide, vinorelbine, vinblastine, vindesine, glucocorticoids, histamine receptor antagonists, angiogenesis inhibitors, radiation, radiosensitizers, anthracyclines, vinca alkaloids, taxanes such as paclitaxel and docetaxel, cell cycle inhibitors such as cyclin-dependent kinase inhibitors, monoclonal antibodies against other tumor antigens, conjugates of monoclonal antibodies and toxins, T-cell adjuvants, bone marrow transplantation, or antigen-presenting cells such as dendritic cell therapy.

[0149] In other embodiments, the present application also embodies methods of treating lipid-related disorders, e.g., hypercholesterolemia and hypertriglyceridemia, and / or improving lipid parameters, e.g., total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very-low-density lipoprotein (VLDL) cholesterol, triglycerides, and non-HDL cholesterol, comprising administering a variant IL-10 molecule or a derivatized form thereof (e.g., PEGylated).

[0150] The most effective IL-10 variants or fusion proteins thereof for treating lipid-related diseases or disorders include those with the lowest inhibitory potency against macrophages. Accordingly, the present inventors have shown that modifying the receptor-binding domain through an amino acid substitution at position 31 (associated with an increased interdomain angle of the homodimer) induces the least amount of macrophage response. Specifically, EBV IL-10 with a V31L substitution in SEQ ID NO: 3 has been shown to reduce macrophage responses (see, e.g., Figure 8A, designated DV05, or SEQ ID NO: 55). Accordingly, one particularly preferred embodiment contemplates the use of diabodies and monobodies comprising IL-10 variant molecules with a DV05-based mutation (a substitution at amino acid position 31 of SEQ ID NO: 3 or SEQ ID NO: 55). In a more preferred embodiment, the method for treating lipid-based diseases or disorders utilizes a fusion protein or fusion protein complex comprising SEQ ID NO: 24-25, 50-51, or 45.

[0151] In yet another embodiment of the present application, an IL-10 variant molecule or fusion protein thereof, viral IL-10 (including EBV or CMV IL-10), or wild-type IL-10, any of which may optionally include PEGylation or HESylation, is used in a method of targeting mast cells by reducing mast cell degranulation. In a preferred embodiment, the method of targeting mast cells comprises contacting with viral IL-10 or an IL-10 variant molecule to treat seasonal allergies or acute anaphylactic responses. In another aspect, the IL-10 variant molecule, viral IL-10 (including EBV or CMV IL-10), or wild-type IL-10 is used in a method for reducing IgE responsiveness.

[0152] In yet another embodiment, the IL-10 variant molecules or fusion proteins thereof of the present application are preferably useful in the described methods (e.g., anti-inflammation and / or cancer) when screening patient populations. In one embodiment, patients exhibiting a profile in which there is an elevated or high IFNγ response are most susceptible or ideal for cancer treatment using the IL-10 variant molecules. In another embodiment, patients exhibiting a profile in which there is a decreased or low IFNγ response are most susceptible or ideal for anti-inflammatory treatment using the IL-10 variant molecules.

[0153] The broad scope of this application is best understood with reference to the following examples, which are not intended to limit the application to any specific embodiments. All citations herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0154] As will be understood by those skilled in the art, numerous modifications and variations of this application can be made without departing from its spirit and scope. The specific embodiments described herein are provided by way of example, and this application is not limited by the terms of the appended claims, nor by the full scope of equivalents to which such claims are entitled. Furthermore, all references, patents, and patent applications cited in the foregoing specification are incorporated herein by reference. [Example]

[0155] The following examples are merely illustrative of various embodiments of the present application and should not be construed as limiting the scope of the present application in any way. Example 1

[0156] EBV-IL-10 variants or fusion proteins thereof are constructed by altering the primary sequence using standard molecular biology cloning techniques. The alterations in the primary sequence are designed to alter the affinity of the receptor-binding domain and to open or close the interdomain angle. Receptor affinity can be altered by changing the amino acids at and around positions 31 and / or 75 in the mature secretory sequence. The interdomain angle can be altered, for example, but not limited to, by introducing proline into the non-alpha helical sequences between helices C and D and between D and E. Proline induces a kink in the linear direction of the primary amino acid sequence, potentially altering the interdomain angle induced by the subsequent secondary and tertiary structure of the D and E helices. Similarly, the introduction of an amino acid with a bulky side chain, such as tryptophan, can introduce less significant changes to the linear structure of the primary amino acid backbone and result in less pronounced changes to the secondary and tertiary structure. Example 2

[0157] The following example provides a description of how variant IL-10 molecules or fusion proteins thereof are evaluated in macrophages.

[0158] Human blood is collected from healthy patient populations or patients with inflammatory diseases (e.g., Crohn's disease), and freshly collected buffy coats are processed using standard Ficoll density gradient centrifugation procedures to obtain PBMCs. PBMCs are then enriched for CD14 monocytes using the EasySep™ Human Monocyte Enrichment Kit (Cat. No. 19059, Stem Cell Technologies) according to the manufacturer's instructions. + The cells are subjected to enrichment for monocytic cells, and the enrichment efficiency is assessed by standard flow cytometry.

[0159] Enriched monocytes were cultured at 2 × 10 in RPMI medium supplemented with 5% human serum and PSG. 6Cells are seeded into 24-well plates at 1000 cells / mL / well. Cells are treated with serial dilutions of variant IL-10 molecules (0, 0.1, 1, 10, 100, 1000 ng / mL) for 1 hour in a 37°C / 5% CO2 humidified incubator, followed by exposure to 10 ng / mL LPS (Cat. No. L4391, Sigma-Aldrich) for 12–16 hours. After overnight incubation, supernatants are harvested and proinflammatory cytokines (IL-6, TNFα, IL-1β) are measured either by standard ELISA or using the iQue Screener (Intellicyt).

[0160] In this study, we compared the effects of non-PEGylated EBV-IL10 and non-PEGylated human IL-10 on the immunosuppressive ability of macrophages using the procedures described above. Figures 2A, 2B, and 3A, 3B show that EBV-IL10 retained the ability to suppress the inflammatory cytokines IL-1β and TNFα, indicating that EBV-IL10 can maintain its inflammation-suppressing ability in a manner similar to that of human IL-10, even though it has a different interdomain angle. Example 3

[0161] The following examples demonstrate that variant IL-10 molecules or fusion proteins thereof bind to human CD8 + We provide an explanation of how T cells are assessed.

[0162] Human blood is collected from a healthy patient population or from patients with inflammatory diseases (e.g., Crohn's disease), and freshly collected buffy matter is processed using a standard Ficoll density gradient centrifugation procedure to obtain PBMCs. The PBMCs are then purified using EasySep™ human CD8 + CD8 T cell enrichment kit (catalog no. 19053, Stem Cell Technologies) was used according to the manufacturer's instructions. +The cells were then subjected to T cell enrichment. Enrichment efficiency was assessed by standard flow cytometry. The enriched cells were suspended in AIMV (Thermo Fisher Scientific, Catalog No. 12055083) culture medium. 24-well plates were coated with 10 micrograms / mL anti-CD3 (Cat. No. 16-0039-85, Thermo Fisher Scientific) and 2 micrograms / mL anti-CD28 (Cat. No. 16-0289-85, Thermo Fisher Scientific) for 2 hours by incubation in a 37°C / 5% CO2 humidified cell culture incubator, followed by washing 1-2 times with 1x PBS.

[0163] Enriched CD8 + T cells (3×10 6 Cells / mL / well) are added to anti-CD3 / anti-CD28 coated plates and incubated for 72 hours in a 37°C / 5% CO2 humidified cell culture incubator.

[0164] After 72 hours, cells are harvested, counted, and 100 μl are reseeded into round-bottom 96-well plates (2×10 cells / well) in the presence or absence of serial dilutions of variant IL-10 molecules (0, 0.1, 1, 10, 100, 1000 ng / mL, added at 100 μL / well) or control samples. Tests are performed in triplicate. Cells are incubated with the variant IL-10 molecules or their fusion proteins for 72 hours in a 37°C / 5% CO2 humidified incubator. After 72 hours, cells are harvested, washed, and reseeded into new round-bottom 96-well plates in the presence of soluble anti-CD3 (catalog number 16-0039-85, Thermo Fisher Scientific) for 4 hours in a 37°C / 5% CO2 humidified incubator.

[0165] In this study, CD8 +The effects of non-PEGylated EBV-IL10 and non-PEGylated human IL-10 on T cell stimulation are compared. Figures 2C and 3C show that EBV-IL10 exhibited attenuated levels of IFNγ (a measure of T cell stimulation) compared to human IL-10. This indicates the ability to modulate T cell stimulation by altering the interdomain angle. Figures 4A and 4B show that half of the treated donors exhibited the desired full anti-inflammatory effect, while half did not. The variant selected for development demonstrated the response of donor 1, complete suppression of inflammatory cytokine secretion by macrophage cells in response to LPS, and activation of CD8 + This would mimic the lack of IFNγ induction from T cells. Donor 2 exhibited the same suppression of inflammatory cytokine secretion by monocytes / macrophages as donor 1, but only shifted the curve and maximum activation of T cell IFNγ secretion to the right. IL-10 variant molecules that alter receptor affinity and interdomain angle should also reduce T cell activation in patients, similar to donor 2. Example 4

[0166] Human monocytes / macrophages, T cells, and murine MC / 9 cells purchased from ATCC were cultured as described above and their responses to mono- or di-N-terminal 5 kDa PEGylated EBV-IL10 were assessed. PEGylation of EBV-IL10 resulted in only a slight reduction in macrophage responses to LPS (Figure 5B), but almost complete suppression of IFNγ induction from stimulated T cells (Figure 5C). Similarly, PEGylation of EBV-IL10 nearly abolished its stimulatory effect on MC / 9 cells (Figure 5A).

[0167] Various forms of EBV-IL-10 variant diabodies using anti-CD3α and anti-EGFR VH and VL regions were tested using an MC / 9 cell proliferation assay. The EBV-10 variant moieties included D:DV05 (EBV IL-10 with a V31L mutation), D:DV06 (EBV IL-10 with an A75I mutation), and D:DV07 (EBV IL-10 with V31L and A75I mutations). In addition, the DV07 diabody, which contains anti-HIV and anti-Ebola VH and VL regions, was also tested. The various variant diabody forms were compared with human IL-10 and EBV IL-10. The results are shown in Figure 15.

[0168] Other forms of EBV-IL-10 fusion proteins were also tested in vitro. In particular, DhivDebo:DV06 (SEQ ID NOs: 26 and 27) and DmadcamDebo:DV06 (SEQ ID NOs: 41 and 42) were compared to human IL-10 in the macrophage and T cell response assays described herein. The results are provided in Figures 20A and 20B. Example 5

[0169] The following examples provide representative protocols for testing IL-10 and IL-10 variant molecules and their fusion proteins in in vivo tumor models. All in vivo studies are performed in accordance with standard operating procedures and established guidelines approved by the Institutional Animal Care and Use Committee ("IACUC").

[0170] Eight week old female Balb / C mice are purchased, quarantined for one week and maintained on normal food and water with bedding changed once a week on a standard 24 hour light / dark cycle.

[0171] CT26 tumor cells (2 × 10 5 The CT26 tumors (average size 50-150 mm) were suspended in Hank's buffered saline and implanted subcutaneously into 8-week-old mice. 3Wild-type Balb / C mice bearing IL-10 (Envigo) or B cell knockout (Jackson) mice are treated subcutaneously (scruff of the neck) for 10 days with IL-10 or IL-10 variant molecules or their fusion proteins (e.g., EBV IL-10 variant molecules with two receptor-binding substitutions, DV07, covalently linked to VH and VL derived from two different antibodies or diabodies (Figure 8C)) at 0.4 and 0.2 mg / kg three times a week (q3w) and 0.2 and 0.1 mg / kg daily (qd) for 5 days with a 2-day rest period. Tumor length and width are measured with electronic calipers every 3 days, and tumor volume is calculated ((L × W 2 ) / 2)). B cells are depleted in wild-type mice by intravenous (iv) administration of 200 μg / mouse of anti-mouse CD20. The results of one such study are provided in FIG. 16, which used an IL-10 variant molecule designated D:DV07, an IL-10 variant with V31L and A75I mutations that contains variable regions derived from anti-CD3α and anti-EGFR.

[0172] Figures 17A and 17B compare two formats of an IL-10 variant fusion protein (i.e., an IL-10 variant containing both the V31L and A75I mutations, DV07), represented by Figures 9C (large format) and 9f (small format), in an in vivo tumor model. The fusion protein is a non-targeting fusion protein and contains VH and VL regions derived from an anti-HIV antibody and an anti-Ebola antibody (large format), as well as VH and VL regions derived from an anti-Ebola antibody. A dosing study tested the effect of the small format non-targeting IL-10 fusion protein administered for 5 days on, 2 days off (Figure 17A) compared with pegylated recombinant human IL-10 (0.75 mg / kg daily). Additionally, a dosing study examined the effects of large and small forms of the untargeted IL-10 fusion protein (Figure 17B) administered three times per week compared with pegylated IL-10 (0.75 mg / kg daily).

[0173] Studies using small and large formats of IL-10 fusion proteins with tumor-targeting capabilities (i.e., an IL-10 variant, DV07, containing both the V31L and A75I mutations) were also tested in vivo. Figure 18A shows results obtained with daily administration of various targeted IL-10 variant fusion proteins, comparing the large format (DegfDebo:DV07) and small format (Degf:DV07) with the small format non-targeted (Debo:DV07) IL-10 fusion protein and pegylated IL-10. Figure 18B shows results obtained with three weekly administrations of various large format targeted IL-10 variant fusion proteins, comparing various doses (1 mg / kg and 0.25 mg / kg) of the large format (DegfDebo:DV07) with the small format non-targeted (DhDe:DV07) IL-10 fusion protein and pegylated IL-10. Figure 18C shows results obtained with three weekly administrations of various small format targeted IL-10 variant fusion proteins, comparing various doses (1 mg / kg and 0.25 mg / kg) of the small format (Degf:DV07) with the small format non-targeted (Debo:DV07) IL-10 fusion protein and pegylated IL-10. Example 6

[0174] The following example provides a representative protocol for testing IL-10 and IL-10 variant molecules and their fusion proteins in an in vivo cholesterol model. All in vivo studies were performed in accordance with standard operating procedures and established guidelines approved by the IACUC.

[0175] Eight week old female C57BL / 6J mice are purchased from a suitable supplier, quarantined for one week and maintained on normal food and water with bedding changed once per week on a standard 24 hour light / dark cycle.

[0176] Eight-week-old female C57BL / 6J mice from Jackson Laboratories are fed a high-fat diet (Envigo) for three weeks. Before treatment with IL-10 or an IL-10 variant or its fusion protein (e.g., an EBV IL-10 variant containing a single substitution at amino acid position 31 (V31L) of SEQ ID NO: 3 linked to a diabody (D:DV05 EBV IL-10 variant)), plasma samples are obtained by retro-orbital bleeding from each mouse. Mice are subcutaneously treated for two weeks with 0.4 and 0.2 mg / kg three times a week (q3w) and 0.2 and 0.1 mg / kg, with five days of treatment followed by two days of rest per week (qd). Animals are treated for two weeks, and a final blood draw is performed, after which pre- and post-dose plasma cholesterol concentrations are quantified. The day before treatment begins, B cells are depleted by intravenous (iv) administration of 200 μg / mouse of anti-mouse CD20. The results of one such study are provided in Figures 19A and 19B. Example 7

[0177] The following example provides a representative protocol for testing IL-10 and IL-10 variant molecules and their fusion proteins in an in vivo dextran sulfate sodium ("DSS") inflammation model. All in vivo studies are performed in accordance with standard operating procedures and established guidelines approved by the IACUC.

[0178] Eight-week-old female Balb / C mice are purchased from a suitable supplier, quarantined for one week, and maintained on normal food and water in a standard 24-hour light / dark cycle with bedding changed once a week. B cell knockout (Jackson) mice are given 4% DSS in water ad libitum for six days, after which they are given normal water. On the fifth day, mice are treated subcutaneously (scruff of the neck) with IL-10 or IL-10 variants or their fusion proteins (e.g., EBV IL-10 variants containing a single substitution at amino acid position 75 (A75I) of SEQ ID NO: 3 linked to a diabody (D:DV06 EBV IL-10 variant)) at 0.4 and 0.2 mg / kg three times a week (q3w), or 0.2 and 0.1 mg / kg daily (qd), for five days on, two days off. Mice are evaluated daily for the following: 1.) Weight 2.) Stool occult blood 3.) Visible bleeding 4.) Stool consistency

[0179] The Disease Activity Index is determined by combining the following scores: 1. Weight loss 2. Stool consistency 3. Bleeding (divide by 3)

[0180] Each score is determined as follows: weight change (0: less than 1%, 1: 1-5%, 2: 5-10%, 3: 10-15%, 4: more than 15%), bloody stool (0: negative, 2: positive), visible bleeding (4), and stool consistency (0: normal, 2: loose stool, 4: diarrhea). List of Preferred Embodiments

[0181] 1. An EBV-IL10 variant protein comprising one or more amino acid additions, deletions, and / or substitutions that exhibit an altered interdomain angle and / or altered affinity for its cognate receptor when compared to wild-type Epstein-Barr virus IL-10 (EBV-IL10), wherein the altered interdomain angle modulates the angle of binding to the cognate receptor upon dimerization.

[0182] 2. The EBV-IL10 protein of the preceding embodiment, wherein the one or more amino acid additions, deletions, and / or substitutions are located in the IL-10 receptor binding domain.

[0183] 3. The EBV-IL10 protein of any of the preceding embodiments, wherein the one or more amino acid additions, deletions, and / or substitutions are located within alpha helix A and / or helix D.

[0184] 4. The EBV-IL10 protein of any of the preceding embodiments, wherein the one or more amino acid additions, deletions, and / or substitutions are present in the linking domain of EBV-IL10.

[0185] 5. The EBV-IL10 protein of any of the preceding embodiments, wherein the one or more amino acid additions, deletions, and / or substitutions are located within the DE loop of EBV-IL10.

[0186] 6. The EBV-IL10 protein of any of the preceding embodiments, wherein the one or more amino acid additions, deletions, and / or substitutions are located within a 12 amino acid linker region found between alpha helix D and alpha helix E or between alpha helix C and alpha helix D, preferably a proline addition or substitution within the 12 amino acid linker region.

[0187] 7. The EBV-IL10 protein of any of the preceding embodiments, wherein the altered affinity for the cognate receptor comprises one or more amino acid additions, deletions, and / or substitutions in the IL-10 receptor binding domain.

[0188] 8. The EBV-IL10 protein of any of the preceding embodiments, further comprising one or more amino acid additions, deletions, and / or substitutions located within alpha helix A and / or alpha helix D.

[0189] 9. The EBV-IL10 protein of any of the preceding embodiments, further comprising one or more amino acid additions, deletions, and / or substitutions in the IL-10 receptor binding domain.

[0190] 10. The EBV-IL10 protein of any of the preceding embodiments, further comprising one or more amino acid additions, deletions, and / or substitutions located within alpha helix A and / or alpha helix D.

[0191] 11. The EBV-IL10 protein of any of the preceding embodiments, wherein the one or more amino acid additions, deletions, and / or substitutions are at amino acid positions 31 and / or 75 of SEQ ID NO:3.

[0192] 12. A monomeric recombinant protein comprising six alpha helices numbered A-F that can form homodimers with identical monomeric proteins, wherein alpha helices D and E are connected by an interchain amino acid linker, and the linker has been modified by the addition, deletion, or substitution of at least one amino acid that changes the intermolecular angle of the protein when homodimerized.

[0193] 13. The recombinant protein of the preceding embodiment, wherein the protein is a viral-derived protein.

[0194] 14. The recombinant protein of any of the previous embodiments, wherein the virus is Epstein-Barr virus (EBV).

[0195] 15. The recombinant protein of any of the previous embodiments, wherein a homodimer formed between two identical monomeric proteins forms a specific angle of interaction with its cognate receptor.

[0196] 16. The recombinant protein of any of the previous embodiments, wherein the angle of interaction is greater than that of the native wild-type protein.

[0197] 17. The recombinant protein of any of the preceding embodiments, wherein the angle of interaction formed upon homodimerization results in the protein having high affinity for its cognate receptor.

[0198] 18. The recombinant protein of any of the previous embodiments, wherein the angle of interaction formed upon homodimerization results in the protein having low affinity for its cognate receptor.

[0199] 19. The recombinant protein of any of the previous embodiments, wherein the angle of interaction is less than that of the native wild-type protein.

[0200] 20. The recombinant protein of any of the previous embodiments, wherein the angle of interaction results in the protein having high affinity for its cognate receptor.

[0201] 21. The recombinant protein of any of the previous embodiments, wherein the angle of interaction results in the protein having low affinity for its cognate receptor.

[0202] 22. The recombinant protein of any of the previous embodiments, wherein the monomeric protein is interleukin-10.

[0203] 23. The recombinant protein of any of the previous embodiments, wherein the monomeric protein is EBV-IL10.

[0204] 24. The recombinant protein of any of the previous embodiments, wherein the angle of the protein is conferred by a modification to the linker that results in an angle of interaction with the cognate receptor.

[0205] 25. A recombinant variant Epstein-Barr virus IL-10 (EBV-IL10) protein, comprising at least one amino acid addition, deletion, or substitution to the linker region between alpha helices D and E of EBV-IL10 and / or the receptor binding region of EBV-IL10.

[0206] 26. The recombinant protein of the preceding embodiment, wherein the variant EBV-IL10 protein interacts with an identical protein to result in a homodimer having an altered angle of interaction with its cognate receptor and / or an altered inter-homodimer angle.

[0207] 27. The recombinant protein of any of the previous embodiments, wherein the variant EBV-IL10 protein forms an angle of interaction and / or an altered inter-homodimer angle greater than the wild-type EBV-IL10 protein.

[0208] 28. The recombinant protein of any of the preceding embodiments, wherein the variant EBV-IL10 protein forms an angle of interaction and / or an altered inter-homodimer angle that is lower than the wild-type EBV-IL10 protein.

[0209] 29. The recombinant protein of any of the preceding embodiments, wherein the angle of interaction formed upon homodimer formation results in a variant EBV-IL10 protein with increased affinity for its cognate receptor.

[0210] 30. The recombinant protein of any of the preceding embodiments, wherein the angle of interaction formed upon homodimer formation results in the variant EBV-IL10 protein having reduced affinity for its cognate receptor.

[0211] 31. The recombinant protein of any of the previous embodiments, wherein the angle of interaction results in increased affinity for its cognate receptor.

[0212] 32. The recombinant protein of any of the previous embodiments, wherein the angle of interaction results in a reduced affinity for its cognate receptor.

[0213] 33. An isolated, recombinant polynucleotide encoding a protein according to any of the preceding embodiments.

[0214] 34. An isolated, recombinant polynucleotide encoding a protein according to any of the preceding embodiments.

[0215] 35. A vector comprising a nucleic acid encoding a protein according to any of the preceding embodiments.

[0216] 36. A host cell comprising a polynucleotide according to any of the preceding embodiments.

[0217] 37. A method of treating or preventing inflammation in a subject, comprising administering to the subject a therapeutically effective amount of a variant protein of any of the preceding embodiments.

[0218] 38. The method of the preceding embodiment, wherein the angle of the altered variant protein is less than that of wild-type EBV-IL10.

[0219] 39. The method of any of the preceding embodiments, wherein the variant protein binds to the IL10 receptor with intermediate affinity when compared to wild-type EBV-IL10.

[0220] 40. The method of any of the preceding embodiments, wherein the inflammation is inflammatory bowel disease (IBD), Crohn's disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), psoriasis, rheumatoid arthritis, acute anaphylactic shock, and / or seasonal allergies.

[0221] 41. A method of treating or preventing an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount of a variant protein of any of the preceding embodiments.

[0222] 42. The method of the preceding embodiment, wherein the angle of the altered variant protein is less than that of wild-type EBV-IL10.

[0223] 43. The method of any of the preceding embodiments, wherein the variant protein binds to the IL10 receptor with intermediate affinity when compared to wild-type EBV-IL10.

[0224] 44. A method of treating or preventing IBD or Crohn's disease in a subject, comprising administering to the subject a therapeutically effective amount of a variant protein of any of the preceding embodiments.

[0225] 45. The method of any preceding embodiment, wherein the angle of the altered variant protein is less than that of wild-type EBV-IL10.

[0226] 46. ​​The method of any of the preceding embodiments, wherein the variant protein binds to the IL10 receptor with intermediate affinity when compared to wild-type EBV-IL10.

[0227] 47. A method for treating or preventing nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH) in a subject, comprising administering to the subject a therapeutically effective amount of the variant protein of claim 1.

[0228] 48. The method of any preceding embodiment, wherein the angle of the altered variant protein is less than that of wild-type EBV-IL10.

[0229] 49. The method of any of the preceding embodiments, wherein the variant protein binds to the IL10 receptor with intermediate affinity when compared to wild-type EBV-IL10.

[0230] 50. A method of treating or preventing cancer in a subject, comprising administering to the subject a therapeutically effective amount of a variant protein of any of the preceding embodiments.

[0231] 51. The method of the preceding embodiment, wherein the angle of the altered variant protein exceeds that of wild-type EBV-IL10.

[0232] 52. The method of any of the preceding embodiments, wherein the variant protein binds to the IL10 receptor with increased affinity when compared to wild-type EBV-IL10.

[0233] 53. An engineered fusion protein comprising at least one monomer of IL-10 or an IL-10 variant molecule conjugated to a first end of the fusion protein, at least one cytokine or a monomer thereof conjugated to a second end of the fusion protein, and a linker or spacer; a linker or spacer connects the first and second ends; Engineered fusion proteins.

[0234] 54. The fusion protein of the preceding embodiment, wherein the linker or spacer is an antibody constant region.

[0235] 55. The fusion protein of any of the previous embodiments, wherein the constant region is derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE.

[0236] 56. The fusion protein of any of the previous embodiments, wherein the linker or spacer further comprises at least two interchain disulfide bonds.

[0237] 57. The fusion protein of any of the previous embodiments, wherein the linker or spacer is an scFv, a diabody, or a fragment thereof.

[0238] 58. The fusion protein of any of the previous embodiments, wherein the constant region is a heavy chain constant (CH) region 1, CH2, CH3, or any combination thereof.

[0239] 59. The fusion protein of any of the preceding embodiments, wherein at least one IL-10 or IL-10 variant molecule is conjugated to the N-terminus, the C-terminus, or both of the fusion protein.

[0240] 60. The fusion protein of any of the preceding embodiments, wherein the at least one cytokine conjugated to the other end comprises IL-10, an IL-10 variant molecule, IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferon-α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or any combination thereof.

[0241] 61. The fusion protein of any of the preceding embodiments, wherein the fusion protein comprises two IL-10 or IL-10 variant molecules conjugated to the N-terminus of the fusion protein, and two IL-10 or IL-10 variant molecules conjugated to the C-terminus of the fusion protein.

[0242] 62. The fusion protein of any of the previous embodiments, wherein the fusion protein comprises two IL-10 or IL-10 variant molecules conjugated to the N-terminus of the fusion protein, and at least one IL-2 molecule conjugated to the C-terminus of the fusion protein.

[0243] 63. The fusion protein of any of the preceding embodiments, wherein the C-terminus further comprises IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferon-α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13.

[0244] 64. The fusion protein of any of the previous embodiments, wherein the fusion protein comprises two IL-10 or IL-10 variant molecules conjugated to the N-terminus of the fusion protein, and at least one IL-15 molecule conjugated to the C-terminus of the fusion protein.

[0245] 65.The C terminus is IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G- A fusion protein v further comprising CSF, interferon-α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13.

[0246] 66. The fusion protein of any of the previous embodiments, wherein the fusion protein comprises two IL-10 or IL-10 variant molecules conjugated to the N-terminus of the fusion protein, and at least one IL-2 molecule conjugated to the C-terminus of the fusion protein.

[0247] 67. The fusion protein of any of the preceding embodiments, wherein the C-terminus further comprises IL-10, an IL-10 variant molecule, IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferon-α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13.

[0248] 68. The fusion protein of any of the previous embodiments, wherein the fusion protein is made on a single-chain variable fragment (scFv) scaffold.

[0249] 69. The fusion protein of any of the previous embodiments, wherein the fusion protein is made on a diabody scaffold.

[0250] 70. The fusion protein of any of the previous embodiments, wherein the fusion protein is made on a Fab scaffold.

[0251] 71. The fusion protein forms a complex with another fusion protein having at least one monomer of IL-10 or an IL-10 variant molecule conjugated to a first end of the fusion protein, at least one cytokine or a monomer thereof conjugated to a second end of the fusion protein, and a linker or spacer; a linker or spacer connects the first and second ends; 1. The fusion protein of any of the preceding embodiments.

[0252] 72. A method of treating cancer in a subject in need thereof, comprising administering to the subject an engineered fusion protein of any of the preceding embodiments.

[0253] 73. A method of treating or preventing IBD or Crohn's disease, comprising administering to a subject an engineered fusion protein of any of the preceding embodiments.

[0254] 74. A method of treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a subject, comprising administering to the subject an engineered fusion protein of any of the preceding embodiments.

[0255] 75. A method of activating CD8 positive T cells, comprising administering an engineered fusion protein according to any of the preceding embodiments.

[0256] 76. The method of any of the preceding embodiments, wherein administering is in vitro administering.

[0257] 77. The method of any of the preceding embodiments, wherein administering is in vivo administration to a subject in need thereof, and the subject has been diagnosed with cancer, IBD, or Crohn's disease, or NAFLD or NASH.

[0258] 78. The method of any of the previous embodiments, wherein the fusion protein comprises an IL-10 or IL-10 variant molecule at a first end of the fusion protein, and IL-2 and / or IL-15 at a second end of the fusion protein.

[0259] 79. A method of treating cancer in a subject in need thereof, comprising administering to the subject a bispecific T cell binding agent (BITE) and IL-10, an IL-10 variant molecule, or an engineered fusion protein comprising IL-10 or an IL-10 variant molecule.

[0260] 80. The method of the preceding embodiment, wherein the engineered fusion protein comprises at least one IL-10 or IL-10 variant molecule conjugated to a first end of the fusion protein, at least one cytokine conjugated to a second end of the fusion protein, and a linker or spacer, wherein the linker or spacer connects the first and second ends.

[0261] 81. The method of any of the preceding embodiments, wherein the IL-10, IL-10 variant molecule, or engineered fusion protein comprising IL-10 or an IL-10 variant molecule increases and sustains T cell receptor complex (CD3) signaling.

[0262] 82. A method of treating or preventing inflammation in a subject, comprising administering to the subject a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

[0263] 83. The method of the preceding embodiment, wherein the nucleotide sequence is DNA, RNA, or an engineered variant thereof.

[0264] 84. The method of any of the preceding embodiments, wherein the nucleotide sequence is a nucleoside-linked mRNA or a modified mRNA.

[0265] 85. The method of any of the preceding embodiments, wherein the nucleotide sequence is capable of in vivo expression of the variant IL-10 molecule in a cell, tissue, or organism.

[0266] 86. The method of any of the preceding embodiments, wherein the nucleotide sequence is delivered to a cell, tissue, or organism by a cell-penetrating peptide, a hydrophobic moiety, an electrostatic complex, a liposome, a ligand, a liposomal nanoparticle, a lipoprotein (preferably HDL or LDL), a folate-targeted liposome, an antibody (e.g., folate receptor, transferrin receptor), a targeting peptide, or by an aptamer.

[0267] 87. A method of treating or preventing an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

[0268] 88. The method of the preceding embodiment, wherein the nucleotide sequence is DNA, RNA, or an engineered variant thereof.

[0269] 89. The method of any of the preceding embodiments, wherein the nucleotide sequence is a nucleoside-linked mRNA or a modified mRNA.

[0270] 90. The method of any of the preceding embodiments, wherein the nucleotide sequence is capable of in vivo expression of the variant IL-10 molecule in a cell, tissue, or organism.

[0271] 91. The method of any of the preceding embodiments, wherein the nucleotide sequence is delivered to a cell, tissue, or organism by a cell-penetrating peptide, a hydrophobic moiety, an electrostatic complex, a liposome, a ligand, a liposomal nanoparticle, a lipoprotein (preferably HDL or LDL), a folate-targeted liposome, an antibody (e.g., folate receptor, transferrin receptor), a targeting peptide, or by an aptamer.

[0272] 92. A method of treating or preventing IBD or Crohn's disease in a subject, comprising administering to the subject a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

[0273] 93. The method of the preceding embodiment, wherein the nucleotide sequence is DNA, RNA, or an engineered variant thereof.

[0274] 94. The method of any of the preceding embodiments, wherein the nucleotide sequence is a nucleoside-linked mRNA or a modified mRNA.

[0275] 95. The method of any of the preceding embodiments, wherein the nucleotide sequence is capable of in vivo expression of the variant IL-10 molecule in a cell, tissue, or organism.

[0276] 96. The method of any of the preceding embodiments, wherein the nucleotide sequence is delivered to a cell, tissue, or organism by a cell-penetrating peptide, a hydrophobic moiety, an electrostatic complex, a liposome, a ligand, a liposomal nanoparticle, a lipoprotein (preferably HDL or LDL), a folate-targeted liposome, an antibody (e.g., folate receptor, transferrin receptor), a targeting peptide, or by an aptamer.

[0277] 97. A method of treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a subject, comprising administering to the subject a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

[0278] 98. The method of any of the preceding embodiments, wherein the nucleotide sequence is DNA, RNA, or an engineered variant thereof.

[0279] 99. The method of any of the preceding embodiments, wherein the nucleotide sequence is a nucleoside-linked mRNA or a modified mRNA.

[0280] 100. The method of any of the preceding embodiments, wherein the nucleotide sequence is capable of in vivo expression of the variant IL-10 molecule in a cell, tissue, or organism.

[0281] 101. The method of any of the preceding embodiments, wherein the nucleotide sequence is delivered to a cell, tissue, or organism by a cell-penetrating peptide, a hydrophobic moiety, an electrostatic complex, a liposome, a ligand, a liposomal nanoparticle, a lipoprotein (preferably HDL or LDL), a folate-targeted liposome, an antibody (e.g., folate receptor, transferrin receptor), a targeting peptide, or by an aptamer.

[0282] 102. A fusion protein comprising a monomeric IL-10 molecule or a variant thereof linked to two variable regions derived from at least two different antibodies, wherein the two variable regions are configured as a heavy chain variable (VH) region derived from a first antibody linked to a light chain variable (VL) region derived from a second antibody, or a VL derived from a first antibody linked to a VH derived from a second antibody.

[0283] 103. The fusion protein of any of the previous embodiments, wherein the monomeric IL-10 molecule or variant thereof comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

[0284] 104. The fusion protein of any of the previous embodiments, wherein the monomeric IL-10 molecule or variant thereof comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0285] 105. The fusion protein of any of the previous embodiments, wherein the monomeric IL-10 molecule or variant thereof is the Epstein-Barr virus (EBV) IL-10 homolog of SEQ ID NO: 3.

[0286] 106. The fusion protein of any of the previous embodiments, wherein the EBV IL-10 homologue comprises an amino acid substitution at position 31, 75, or both.

[0287] 107. The fusion protein of any of the previous embodiments, wherein the EBV-IL-10 homolog comprises an amino acid substitution at position 31.

[0288] 108. The fusion protein of any of the previous embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 75.

[0289] 109. The fusion protein of any of the previous embodiments, wherein the EBV-IL-10 homologue comprises amino acid substitutions at positions 31 and 75.

[0290] 110. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises a V31L amino acid substitution.

[0291] 111. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises an A75I amino acid substitution.

[0292] 112. The fusion protein of any of the previous embodiments, wherein the EBV-IL-10 homolog comprises V31L and A75I amino acid substitutions.

[0293] 113. The fusion protein of any of the previous embodiments, wherein the VH region of the first antibody is derived from an anti-HIV monoclonal antibody and the VL region of the second antibody is derived from an anti-Ebola monoclonal antibody.

[0294] 114. The fusion protein of any of the preceding embodiments, wherein the VH region of the first antibody is derived from an anti-Ebola monoclonal antibody, and the VL region of the second antibody is derived from an anti-HIV monoclonal antibody.

[0295] 115. The fusion protein of any of the previous embodiments, wherein the fusion protein is an amino acid sequence selected from SEQ ID NOs: 24-28, 29, 33-51, 61, 63, 65, or 67.

[0296] 116. The fusion protein of any of the previous embodiments, wherein the fusion protein is a diabody.

[0297] 117. A fusion protein comprising: (a) a VH region of a first antibody linked at its carboxy terminus to the amino terminus of a VL region of a second antibody, which is subsequently linked to the amino terminus of a monomer of IL-10 or a variant thereof; or (b) the IL-10 molecule or variant thereof is linked at its carboxy terminus to the amino terminus of a VH region of a second antibody, which is in turn linked to the amino terminus of a VL region of a first antibody.

[0298] 118. The fusion protein of any of the previous embodiments, wherein components (a) and (b) together form a diabody complex.

[0299] 119. The fusion protein of any of the previous embodiments, further comprising a linker between the VH and VL regions.

[0300] 120. The fusion protein of any of the previous embodiments, wherein the amino acid sequence is selected from SEQ ID NOs: 24-28, 29, 33-53, 61, 63, 65, 67.

[0301] 121. The fusion protein of any of the previous embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in a subject.

[0302] 122. An immunoconjugate complex comprising: i) a first fusion protein comprising, at its amino terminus, the heavy chain variable region (VH) of a first antibody linked to the light chain variable region (VL) of a second antibody, which is further linked to a monomer of IL-10 or a variant thereof; and ii) a second fusion protein comprising, at its amino terminus, a monomer of IL-10 or a variant thereof linked to the VH of the second antibody, which is further linked to the VL of the first antibody, wherein the VH and VL of the first and second antibodies associate as a diabody and the IL-10 monomers form a functional dimeric IL-10 molecule.

[0303] 123. The immunoconjugate complex of any of the preceding embodiments, wherein the IL-10 monomer comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

[0304] 124. The immunoconjugate complex of any of the preceding embodiments, wherein the IL-10 molecule comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0305] 125. The immunoconjugate complex of any of the preceding embodiments, wherein the IL-10 monomer is the Epstein-Barr virus (EBV) IL-10 homolog of SEQ ID NO:3.

[0306] 126. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV IL-10 homolog comprises an amino acid substitution at position 31, 75, or both.

[0307] 127. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 31.

[0308] 128. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 75.

[0309] 129. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises amino acid substitutions at positions 31 and 75.

[0310] 130. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises a V31L amino acid substitution.

[0311] 131. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises an A75I amino acid substitution.

[0312] 132. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises V31L and A75I amino acid substitutions.

[0313] 133. The immunoconjugate complex of any of the preceding embodiments, wherein the first antibody is derived from an anti-HIV monoclonal antibody and the second antibody is derived from an anti-Ebola monoclonal antibody.

[0314] 134. The immunoconjugate complex of any of the previous embodiments, wherein the first fusion protein is an amino acid sequence selected from SEQ ID NOs: 24, 26, 28, 35, 38, 41, 46, 48, or 50.

[0315] 135. The immunoconjugate complex of any of the previous embodiments, wherein the second fusion protein is an amino acid sequence selected from SEQ ID NOs: 25, 27, 29, 36, 39, 42, 47, 49, or 51.

[0316] 136. The immunoconjugate complex of any of the preceding embodiments, further comprising a linker between the VH and VL regions.

[0317] 137. The immunoconjugate complex of any of the previous embodiments, wherein the IL-10 monomer in the first fusion protein is linked by its amino terminus to the VL region.

[0318] 138. The immunoconjugate complex of any of the preceding embodiments, wherein the IL-10 monomer in the second fusion protein is linked by its carboxy terminus to the VH region.

[0319] 139. The immunoconjugate complex of any of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in a subject.

[0320] 140. A diabody comprising a first peptide chain comprising a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and a monomeric IL-10, and a second peptide chain comprising a VH and VL from a second antibody and a monomeric IL-10 molecule, wherein the VH region of the first antibody associates with the VL region of the first antibody and the VH region of the second antibody associates with the VL region of the second antibody, thereby enabling the monomeric IL-10 molecules of each peptide chain to form a functional IL-10 dimer.

[0321] 141. The diabody of any of the previous embodiments, wherein the monomeric IL-10 comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

[0322] 142. The diabody of any of the previous embodiments, wherein the monomeric IL-10 molecule comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0323] 143. The diabody of any of the previous embodiments, wherein the monomeric IL-10 molecule is the Epstein-Barr Virus (EBV) IL-10 homolog of SEQ ID NO:3.

[0324] 144. The diabody of any of the preceding embodiments, wherein the EBV IL-10 homolog comprises an amino acid substitution at position 31, 75, or both.

[0325] 145. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises an amino acid substitution at position 31.

[0326] 146. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises an amino acid substitution at position 75.

[0327] 147. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises amino acid substitutions at positions 31 and 75.

[0328] 148. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises a V31L amino acid substitution.

[0329] 149. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises an A75I amino acid substitution.

[0330] 150. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises V31L and A75I amino acid substitutions.

[0331] 151. The diabody of any of the preceding embodiments, wherein the first antibody is derived from an anti-HIV monoclonal antibody and the second antibody is derived from an anti-Ebola monoclonal antibody.

[0332] 152. The diabody of any of the preceding embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NOs: 24, 26, 28, 35, 38, 41, 46, 48, or 50.

[0333] 153. The diabody of any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NOs: 25, 27, 29, 36, 39, 42, 47, 49, or 51.

[0334] 154. The diabody of any of the previous embodiments, further comprising a linker between the VH and VL regions.

[0335] 155. The diabody of any of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in a subject.

[0336] 156. The diabody of any of the previous embodiments, wherein the monomer of IL-10 is linked by its carboxy terminus to the first and second peptide chains.

[0337] 157. The diabody of any of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in a subject.

[0338] 158. An immunoconjugate complex comprising: i) a first fusion protein comprising at its amino terminus a heavy chain variable (VH) region of a first antibody and a monomeric IL-10 molecule linked by its amino terminus; and ii) a second fusion protein comprising at its amino terminus an IL-10 monomer linked to a light chain variable region (VL) of the first antibody, wherein the VH region of the first antibody associates with the VL region of the first antibody, thereby enabling the monomeric IL-10 molecules of each peptide chain to form a functional IL-10 dimer.

[0339] 159. The immunoconjugate complex of any of the preceding embodiments, wherein the IL-10 monomer comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

[0340] 160. The immunoconjugate complex of any of the preceding embodiments, wherein the IL-10 molecule comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0341] 161. The immunoconjugate complex of any of the preceding embodiments, wherein the monomer of IL-10 is the Epstein-Barr virus (EBV) IL-10 homolog of SEQ ID NO:3.

[0342] 162. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV IL-10 homologue comprises an amino acid substitution at position 31, 75, or both.

[0343] 163. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 31.

[0344] 164. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 75.

[0345] 165. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises amino acid substitutions at positions 31 and 75.

[0346] 166. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises a V31L amino acid substitution.

[0347] 167. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises an A75I amino acid substitution.

[0348] 168. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises V31L and A75I amino acid substitutions.

[0349] 169. The immunoconjugate complex of any of the previous embodiments, wherein the VH and VL regions of the first antibody are derived from an anti-epidermal growth factor receptor (EGFR) monoclonal antibody.

[0350] 170. The immunoconjugate complex of any of the preceding embodiments, wherein the first fusion protein further comprises a VL region derived from a second antibody that links the VH region of the first antibody to monomeric IL-10, and wherein the second fusion protein further comprises a VH region derived from the second antibody that links monomeric IL-10 to the VL region.

[0351] 171. The immunoconjugate complex of any of the preceding embodiments, wherein the VH and VL regions of the second antibody are derived from an anti-Ebola monoclonal antibody.

[0352] 172. The immunoconjugate complex of any of the previous embodiments, wherein the variable region is linked to the monomer of IL-10 through a linker.

[0353] 173. The immunoconjugate complex of any of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in a subject.

[0354] 174. The immunoconjugate complex of any of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in a subject.

[0355] 175. A fusion protein comprising the variable light (VL) and variable heavy (VH) regions of a first antibody fused to monomers of IL-10, wherein the IL-10 monomers are directly linked to each other.

[0356] 176. The fusion protein of any of the preceding embodiments, wherein the IL-10 monomers are linked from the carboxy terminus of a first IL-10 monomer to the amino terminus of a second IL-10 monomer.

[0357] 177. The fusion protein of any of the preceding embodiments, wherein the fusion protein comprises the following configuration, from amino terminus to carboxy terminus: a VL region of a first antibody linked to a first IL-10 monomer, which is linked to a second IL-10 monomer, which is linked to a VH region of the first antibody.

[0358] 178. The fusion protein of any of the preceding embodiments, further comprising: a VH region of a second antibody linked to the amino terminus of the VL region of the first antibody; and a VL region of the second antibody linked to the carboxy terminus of the VH region of the first antibody.

[0359] 179. The fusion protein of any of the previous embodiments, wherein the IL-10 monomer comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor, respectively.

[0360] 180. The fusion protein of any of the preceding embodiments, wherein the IL-10 monomers each comprise at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0361] 181. The fusion protein of any of the previous embodiments, wherein the IL-10 monomer is the Epstein-Barr Virus (EBV) IL-10 homolog of SEQ ID NO:3.

[0362] 182. The fusion protein of claim 181, wherein the EBV IL-10 homolog comprises an amino acid substitution at position 31, 75, or both.

[0363] 183. The fusion protein of any of the previous embodiments, wherein the EBV-IL-10 homolog comprises an amino acid substitution at position 31.

[0364] 184. The fusion protein of any of the previous embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 75.

[0365] 185. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises a V31L amino acid substitution.

[0366] 186. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homolog comprises an A75I amino acid substitution.

[0367] 187. The fusion protein of any of the preceding embodiments, wherein the first antibody is an anti-Ebola monoclonal antibody.

[0368] 188. The fusion protein of any of the previous embodiments, wherein the first antibody is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody.

[0369] 189. The fusion protein of any of the preceding embodiments, wherein the first antibody is an anti-Ebola monoclonal antibody and the second antibody is an anti-EGFR monoclonal antibody.

[0370] 190. A method of treating a disease, disorder, or condition in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an Epstein-Barr virus (EBV) IL-10 immunoconjugate complex, wherein the immunoconjugate complex has a molecular weight of about 60 to 155 kDa, the therapeutically effective amount is in the range of about 0.5 micrograms / kilogram to 100 micrograms / kilogram, and the EBV IL-10 portion of the immunoconjugate is derived from SEQ ID NO:3.

[0371] 191. The method of any of the preceding embodiments, wherein the immunoconjugate complex is administered once a month, twice a month, once a week, twice a week, three times a week, or once a day.

[0372] 192. The method of any of the preceding embodiments, wherein the variant EBV IL-10 is a variant comprising at least one amino acid substitution that increases or decreases binding to the IL-10 receptor.

[0373] 193. The method of any of the preceding embodiments, wherein the variant EBV IL-10 comprises an amino acid substitution at position 31, 75, or both of SEQ ID NO:3.

[0374] 194. The method of any of the preceding embodiments, wherein the variant EBV IL-10 comprises a V31L amino acid substitution.

[0375] 195. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises an A75I amino acid substitution.

[0376] 196. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises V31L and A75I amino acid substitutions.

[0377] 197. The method of any of the preceding embodiments, wherein the immunoconjugate complex is a complex of two fusion proteins.

[0378] 198. The method of any of the preceding embodiments, wherein the immunoconjugate complex comprises: i. a first fusion protein comprising, at its amino terminus, the heavy chain variable region (VH) of a first antibody linked to the light chain variable region (VL) of a second antibody, which is further linked to the carboxy terminus of an EBV IL-10 monomer; and ii. a second fusion protein comprising, at its amino terminus, an EBV IL-10 monomer linked to the VH of the second antibody, which is further linked to the VL of the first antibody, wherein the VH and VL of the first and second antibodies associate as a diabody and the EBV IL-10 monomers form a functional dimeric EBV IL-10 molecule.

[0379] 199. The method of any of the preceding embodiments, wherein the first antibody and the second antibody are different antibodies.

[0380] 200. The method of any of the preceding embodiments, wherein the first antibody is an anti-HIV monoclonal antibody and the second antibody is an anti-Ebola monoclonal antibody.

[0381] 201. The method of claim 202, wherein the fusion protein is an amino acid sequence selected from SEQ ID NOs: 24-51.

[0382] 202. The method of claim 202, wherein the first fusion protein is an amino acid sequence selected from SEQ ID NOs: 24, 26, 28, 35, 38, 41, 46, 48, or 50.

[0383] 203. The method of claim 202, wherein the second fusion protein is an amino acid sequence selected from SEQ ID NOs: 25, 27, 29, 36, 39, 42, 47, 49, or 51.

[0384] 204. The method of any of the preceding embodiments, wherein the immunoconjugate complex is a diabody comprising EBV IL-10 monomers fused to either terminus, and the EBV IL-10 monomers are capable of associating into functional EBV IL-10 dimers.

[0385] 205. The method of any of the preceding embodiments, wherein the disease, disorder, or condition is selected from cancer, an inflammatory disease, an autoimmune disease, or cholesterol.

[0386] 206. The method of any of the preceding embodiments, wherein the EBV IL-10 immunoconjugate complex is administered in an amount sufficient to maintain stable IL-10 serum concentrations based on administration at least every 2-3 days.

[0387] 207. The method of any of the preceding embodiments, wherein the immunoconjugate is capable of suppressing TNFα secretion and inducing IFNγ production at concentrations similar to wild-type IL-10.

[0388] 208. The method of any of the preceding embodiments, wherein the EBV IL-10 immunoconjugate complex has activity similar to wild-type IL-10.

[0389] 209. The immunoconjugate complex is (a) the VH region of a first antibody linked at its N-terminus to the VL region of a second antibody, which is linked to the carboxy terminus of an IL-10 molecule; (b) an IL-10 molecule linked to the VH region of a second antibody, which is linked to the VL region of a first antibody; 10. The method of any preceding embodiment, comprising:

[0390] 210. A method of treating cancer in a patient in need thereof, comprising administering to the patient a diabody comprising a first peptide chain having a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and a monomeric IL-10 molecule, and a second peptide chain having a VH and VL from the second antibody and a monomeric IL-10 molecule, wherein the VH region of the first antibody associates with the VL region of the first antibody and the VH region of the second antibody associates with the VL region of the second antibody, thereby enabling the monomeric IL-10 molecules of each peptide chain to form a functional IL-10 dimer.

[0391] 211. The method of any of the preceding embodiments, wherein the monomeric IL-10 is the Epstein-Barr virus (EBV) IL-10 homolog of SEQ ID NO: 3.

[0392] 212. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises an amino acid substitution at position 31 of SEQ ID NO:3.

[0393] 213. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

[0394] 214. The method of any of the preceding embodiments, wherein the VH region of the first antibody is derived from an anti-HIV monoclonal antibody, and the VL region of the second antibody is derived from an anti-Ebola monoclonal antibody.

[0395] 215. The method of any of the preceding embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NOs: 28, 35, 38, 46.

[0396] 216. The method of any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NOs: 29, 36, 39, 47.

[0397] 217. The method of any of the preceding embodiments, further comprising a linker between the VH and VL regions.

[0398] 218. The method of any of the preceding embodiments, wherein VH and VL each comprise one or more amino acid substitutions that reduce antigenicity in a patient.

[0399] 219. The method of any of the preceding embodiments, wherein the first antibody is derived from an anti-HIV monoclonal antibody and the second antibody is derived from an anti-Ebola monoclonal antibody.

[0400] 220. A diabody comprising, from amino terminus to carboxy terminus: (a) a first peptide in which the VH region of a first antibody is linked to the VL region of a second antibody, which is linked to the amino terminus of an IL-10 monomer or a variant thereof; (b) the second peptide comprises an IL-10 monomer linked to the VH region of a second antibody, which is linked to the VL region of the first antibody; 10. The method of any preceding embodiment, wherein the amino acid sequence is formed by two peptide chains having the structure:

[0401] 221. A method of treating cholesterol in a patient in need thereof, comprising the step of administering to the patient a cholesterol-reducing amount of a diabody comprising a first peptide chain having a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and a monomeric IL-10, and a second peptide chain having a VH and VL from the second antibody and a monomeric IL-10 molecule, wherein the VH region of the first antibody associates with the VL region of the first antibody and the VH region of the second antibody associates with the VL region of the second antibody, thereby allowing the monomeric IL-10 molecules of each peptide chain to form a functional IL-10 dimer.

[0402] 222. The method of any of the preceding embodiments, wherein the monomeric IL-10 is the Epstein-Barr virus (EBV) IL-10 homolog of SEQ ID NO: 3.

[0403] 223. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises an amino acid substitution at position 31 of SEQ ID NO:3.

[0404] 224. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

[0405] 225. The method of any of the preceding embodiments, wherein the VH region of the first antibody is derived from an anti-HIV monoclonal antibody and the VL region of the second antibody is derived from an anti-Ebola monoclonal antibody.

[0406] 226. The method of any of the previous embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NO: 24 or 50.

[0407] 227. The method of any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NO: 25 or 51.

[0408] 228. The method of any of the preceding embodiments, further comprising a linker between the VH and VL regions.

[0409] 229. The method of any of the preceding embodiments, wherein VH and VL each comprise one or more amino acid substitutions that reduce antigenicity in a patient.

[0410] 230. The method of any of the preceding embodiments, wherein the first antibody is derived from an anti-HIV monoclonal antibody and the second antibody is derived from an anti-Ebola monoclonal antibody.

[0411] 231. A diabody comprising, from amino terminus to carboxy terminus: (a) the first peptide chain comprises a VH region of a first antibody linked to a VL region of a second antibody, which is linked to the amino terminus of an IL-10 monomer or a variant thereof; and (b) the second peptide comprises an IL-10 monomer or a variant thereof linked to a VH region of a second antibody, which is linked to the VL region of the first antibody. 10. The method of any preceding embodiment, wherein the amino acid sequence is formed by two peptide chains having the structure:

[0412] 232. A method of treating non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD) in a patient in need thereof, comprising administering to the patient an NASH or NAFLD amount of a diabody comprising a first peptide chain having a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and a monomeric IL-10, and a second peptide chain having a VH and VL from the second antibody and a monomeric IL-10 molecule, wherein the VH region of the first antibody associates with the VL region of the first antibody and the VH region of the second antibody associates with the VL region of the second antibody, thereby enabling the monomeric IL-10 molecules of each peptide chain to form a functional IL-10 dimer.

[0413] 233. The method of any of the preceding embodiments, wherein the monomeric IL-10 is the Epstein-Barr virus (EBV) IL-10 homolog of SEQ ID NO: 3.

[0414] 234. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises an amino acid substitution at position 31 of SEQ ID NO:3.

[0415] 235. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

[0416] 236. The method of any of the preceding embodiments, wherein the VH region of the first antibody is derived from an anti-HIV monoclonal antibody and the VL region of the second antibody is derived from an anti-Ebola monoclonal antibody.

[0417] 237. The method of any of the previous embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NO: 24 or 50.

[0418] 238. The method of any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NO: 25 or 51.

[0419] 239. The method of any of the preceding embodiments, further comprising a linker between the VH and VL regions.

[0420] 240. The method of any of the preceding embodiments, wherein VH and VL each comprise one or more amino acid substitutions that reduce antigenicity in a patient.

[0421] 241. The method of any of the preceding embodiments, wherein the first antibody is derived from an anti-HIV monoclonal antibody and the second antibody is derived from an anti-Ebola monoclonal antibody.

[0422] 242. A method of treating inflammation in a patient in need thereof, comprising the step of administering to the patient an anti-inflammatory amount of a diabody comprising a first peptide chain having a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and a monomeric IL-10, and a second peptide chain having a VH and VL from the second antibody and a monomeric IL-10 molecule, wherein the VH region of the first antibody associates with the VL region of the first antibody and the VH region of the second antibody associates with the VL region of the second antibody, thereby allowing the monomeric IL-10 molecules of each peptide chain to form a functional IL-10 dimer.

[0423] 243. The method of any of the preceding embodiments, wherein the monomeric IL-10 is the Epstein-Barr virus (EBV) IL-10 homolog of SEQ ID NO: 3.

[0424] 244. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises an amino acid substitution at position 75 of SEQ ID NO:3.

[0425] 245. The method of any of the preceding embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

[0426] 246. The method of any of the preceding embodiments, wherein the VH region of the first antibody is derived from an anti-HIV monoclonal antibody and the VL region of the second antibody is derived from an anti-Ebola monoclonal antibody.

[0427] 247. The method of any of the preceding embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NO: 26, 41, or 48.

[0428] 248. The method of any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NOs: 27, 42, 49.

[0429] 249. The method of any of the preceding embodiments, further comprising a linker between the VH and VL regions.

[0430] 250. The method of any of the preceding embodiments, wherein VH and VL each comprise one or more amino acid substitutions that reduce antigenicity in a patient.

[0431] 251. The method of any of the preceding embodiments, wherein the first antibody is derived from an anti-HIV monoclonal antibody and the second antibody is derived from an anti-Ebola monoclonal antibody.

[0432] 252. Formula (I-VII) IL10-L 1 -X 1 -L 1 -X 2 -L 1 -IL10 (Formula I), (Z) n -X 1 -L 2 -Y 2 -L 1 -IL10 (Formula II), IL10-L 1 -Y 1 -L 2 -X 2 -(Z) n (Formula III), X 1 -L 2 -X 2 -L 1 -IL10 (Formula IV), IL10-L 1 -X 1 -L 2 -X 2 (Formula V), X 1 -L 1 -IL10 (Formula VI), IL10-L 1 -X2 (Formula VII), or any combination thereof A fusion protein of During the ceremony, "IL-10" is a monomer sequence selected from SEQ ID NO: 1, 3, 14, 18, 15, 19, 16, 20, 55, 57, or 59, more preferably "IL-10" consists of SEQ ID NO: 55, 57, or 59; "L 1 " is a linker of SEQ ID NO: 31 or 54, "L2 " is the linker of SEQ ID NO: 30, "X 1 " is a VH region obtained from a first antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as, but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin; SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola, "X2" is a VL region obtained from the same antibody as X1, "Y1" is a VH region obtained from a second antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as, but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin; SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola; "Y2" is a VL region obtained from the same antibody as Y1, X and Y are derived from the same or different antibodies, "Z" is a cytokine selected from IL-10, an IL-10 variant molecule, IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferon-α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13; "n" is an integer selected from 0 to 2; Fusion proteins.

[0433] 253. The fusion protein of the preceding embodiment, wherein Formulas II and III are capable of forming a fusion protein complex, and wherein IL-10 monomers derived from each of Formulas II and III are capable of forming a functional homodimeric IL-10 or a variant thereof.

[0434] 254. The fusion protein of any of the previous embodiments, wherein Formula II is SEQ ID NO: 24, 26, 28, 41, 48, or 50.

[0435] 255. The fusion protein of any of the previous embodiments, wherein Formula III is SEQ ID NO: 25, 27, 29, 42, 49, or 51.

[0436] 256. The fusion protein of any of the preceding embodiments, wherein the fusion protein complex is formed between SEQ ID NOs: 24 and 25, between 26 and 27, between 28 and 29, between 41 and 42, between 48 and 49, or between 50 and 51.

[0437] 257. The fusion protein of any of the preceding embodiments, wherein Formulas IV and V are capable of forming a fusion protein complex, and wherein IL-10 monomers derived from each of Formulas IV and V are capable of forming functional homodimeric IL-10 or a variant thereof.

[0438] 258. The fusion protein of any of the previous embodiments, wherein formula IV is SEQ ID NO: 35, 38, 46, 48, or 50.

[0439] 259. The fusion protein of any of the previous embodiments, wherein Formula V is SEQ ID NO: 36, 39, 47, 49, or 51.

[0440] 260. The fusion protein of any of the previous embodiments, wherein the fusion protein complex is formed between SEQ ID NOs: 35 and 36, between 38 and 39, between 46 and 47, between 48 and 49, or between 50 and 51.

[0441] 261. The fusion protein of any of the preceding embodiments, wherein Formulas VI and VII are capable of forming a fusion protein complex, and wherein IL-10 monomers derived from each of Formulas VI and VII are capable of forming functional homodimeric IL-10 or a variant thereof.

[0442] 262. The fusion protein of any of the previous embodiments, wherein Formula I is SEQ ID NO: 33-34, 40, 43-44, 45, 52, 53, 61, 63, 65, or 67.

[0443] 263. The fusion protein of any of the previous embodiments, wherein "n" is 1 or more, and Z is IL-2, IL-7, IL-12, IL-15, or any combination thereof.

[0444] 264.Z is X 1 , Y 1

[0023] The fusion protein of any of the preceding embodiments, wherein the nucleotide sequence is conjugated to the N-terminus of the fusion protein.

[0445] 265. A method of treating cancer, comprising administering to a patient in need thereof a composition comprising the fusion protein of any of the preceding embodiments.

[0446] 266. The method of any of the preceding embodiments, wherein the fusion protein is SEQ ID NO: 28-29, 35-36, 38-39, 46-47, 52, 53, 61, 63, 65, or 67.

[0447] 267. The method of any of the preceding embodiments, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID NOs: 28 and 29, between 35 and 36, between 38 and 39, or between 46 and 47.

[0448] 268. The method of any of the preceding embodiments, wherein the composition comprises a fusion protein of SEQ ID NO: 33, 34, 44, 52, or 53, 61, 63, 65, or 67.

[0449] 269. The method of any of the previous embodiments, wherein the fusion protein comprises IL-10 consisting of DV07 of SEQ ID NO: 59.

[0450] 270. A method of treating an inflammatory disease, comprising administering to a patient in need thereof a composition comprising the fusion protein of any of the preceding embodiments.

[0451] 271. The method of any of the preceding embodiments, wherein the fusion protein is SEQ ID NO: 26-27, 41-42, 48, or 49.

[0452] 272. The method of any of the preceding embodiments, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID NOs: 26 and 27, between 41 and 42, between 48 and 49.

[0453] 273. The method of any of the preceding embodiments, wherein the composition comprises a fusion protein of SEQ ID NO: 37, 40, or 43.

[0454] 274. The method of claim 18, wherein the fusion protein comprises IL-10 consisting of DV06 of SEQ ID NO: 57.

[0455] 275. A method of treating a lipid-based disease, comprising administering to a patient in need thereof a composition comprising the fusion protein of any of the preceding embodiments.

[0456] 276. The method of any of the preceding embodiments, wherein the fusion protein is SEQ ID NO: 24-25, 50, or 51.

[0457] 277. The method of any of the previous embodiments, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID NOs: 24 and 25, 50 and 51.

[0458] 278. The method of any of the preceding embodiments, wherein the composition comprises a fusion protein of SEQ ID NO: 45.

[0459] References [ka] [ka] [ka] [ka]

Claims

[Claim 1] The invention described in the specification.

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