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

Abstract

The present application relates to compositions or formulations comprising variant IL-10 molecules, fusion proteins and chimeric proteins thereof, useful for the treatment of cancer, inflammatory diseases or conditions, and autoimmune diseases or conditions.

Description

[0001] This application is a divisional application of PCT application PCT / US2020 / 021498, filed on March 6, 2020, entitled "IL-10 variant molecule and method for treating inflammatory diseases and tumors". The date of entry into the Chinese national phase of the PCT application was November 4, 2021, and the application number was 2020800336707.

[0002] Cross-reference to related applications

[0003] 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 disclosure of each of which is incorporated herein by reference in its entirety.

[0004] introduction

[0005] This application relates to variant forms of interleukin-10 (IL-10) comprising modifications to the IL-10 receptor-binding region and / or the domain responsible for interdomain angles present in the IL-10 molecule. By modifying one or both of these domains in IL-10, the inventors have surprisingly found that the biological function of the resulting IL-10 receptor can be modulated or regulated to elicit specific biological responses. This application also relates to IL-10 or IL-10 variant molecules with extended half-lives, comprising a non-protein-based serum extension portion and a protein-based extension. This application further relates to fusion proteins comprising IL-10 variant molecules. Background Technology

[0006] IL-10 has been described as a cytokine synthesis inhibitor because it can inhibit (i) the secretion of pro-inflammatory cytokines by monocytes / macrophages in response to lipopolysaccharide, and (ii) the secretion of interleukin-2 (IL-2) and CD4+. + T cell proliferation. When viral analogues of IL-10 are discovered and reported to have similar or identical functions to human IL-10, it is speculated that these viral analogues of IL-10 enhance viral virulence by employing the function of inhibitory cytokines found in the human genome.

[0007] By generating IL-10 knockout mice with chronic enterocolitis, further research into the inhibitory role of IL-10 became possible. Data from these mice clearly demonstrate that IL-10 knockout mice exhibit severe inflammation throughout the gastrointestinal tract, primarily mediated by monocytes / macrophages and CD4+. + The chronic inflammatory cytokines secreted by T cells are consistent with the initial findings observed in vitro.

[0008] Overall, these data suggest that IL-10 plays a dominant role in suppressing inflammation. In particular, patients lacking functional IL-10 receptors or the ability to produce IL-10 showed an increased predisposition to inflammation-related gastrointestinal disorders.

[0009] Several clinical trials have been conducted to evaluate the anti-inflammatory function of IL-10 in the context of psoriasis, rheumatoid arthritis, and Crohn's disease. Generally, treatment with recombinant human IL-10 (rHuIL-10) has been found to be safe, but lacks efficacy. In particular, treatment with rHuIL-10 in Crohn's disease patients resulted in an inverse dose-response, where low doses appeared to moderately suppress inflammation, while the inhibitory effect disappeared at high doses. A rigorous analysis of the final Crohn's study showed that patients taking 10 and 20 μg / kg rHuIL-10 exhibited increased serum concentrations of interferon gamma (IFNγ) and neopterin. Interferon gamma is known to exacerbate the pathogenesis of inflammatory bowel disease and Crohn's disease. The data suggest that at high doses, IL-10 treatment induces IFNγ, which in turn exacerbates the inflammatory disease.

[0010] Further analysis of the effects of IL-10 in healthy individuals showed that administration of IL-10 prior to exposure to the pro-inflammatory cytokine lipopolysaccharide (LPS) inhibited the production of pro-inflammatory cytokines. However, administration of rHuIL-10 after LPS exposure enhanced the secretion of pro-inflammatory cytokines. Since LPS is a product of both normal and exogenous gut bacteria in patients with inflammatory bowel disease (IBD), these patients are never "LPS-free" and therefore never in a state where IL-10 treatment could be administered prior to LPS exposure. Therefore, these data suggest that patients with Crohn's disease and other inflammatory diseases will never see the therapeutic benefit of IL-10 treatment.

[0011] Adding to the confusion surrounding the role of IL-10 is the accumulation of data suggesting that it activates the immune system to induce an anti-tumor response. Initial data indicated that IL-10 activates NK cells, but further research revealed that IL-10 treatment targets CD8+ cells. + T cells and IFNγ-dependent mechanisms inhibit tumor growth. Further research revealed that IL-10 treatment inhibits FoxP3. + CD4 + The proliferation of regulatory T cells and the enhancement of Kupffer cell clearance functions suggest that IL-10 is an effective immunostimulant, rather than an inhibitor. Finally, these stimulatory activities were confirmed in clinical studies of cancer patients treated with pegylated IL-10.

[0012] In summary, these data suggest that IL-10 treatment does not provide a therapeutic benefit in patients with autoimmune-related inflammation, indicating that IL-10 is not a pan-immunosuppressant. Consistent with this, treatment of cancer patients with (PEG)IL-10 resulted in effective and therapeutically beneficial immune activation, particularly inducing dose-dependent serum IFNγ, similar to that in Crohn's disease patients treated with IL-10, suggesting that IL-10 is an effective immunostimulant.

[0013] The unresolved question is why viruses acquire the IL-10 sequence. Further analysis of Epstein-Barr virus (EBV-IL10) and cytomegalovirus (CMV-IL10) homologs revealed that these viruses possess a modified natural IL-10 sequence in two main ways. EBV-IL10 appears to retain a similar spatial angle of homodimer interaction leading to a specific angle of ligand-receptor interaction, while its affinity for the IL-10 receptor is significantly reduced. CMV-IL10 exhibits increased affinity for the IL-10 receptor, while the angle of interaction with the IL-10 receptor is significantly altered. Although the EBV-IL10 and CMV-IL10 sequences retain approximately 80% and 27% homology with natural IL-10, respectively, each viral homolog possesses a completely different IL-10 receptor affinity and a different receptor-binding angle, and each viral homolog exhibits anti-inflammatory functions highly similar to those of natural IL-10. Therefore, it remains unclear how affinity for the IL-10 receptor and / or the angle of receptor interaction affect the downstream transduction of subsequent IL-10 signaling.

[0014] Overview of various preferred implementation schemes

[0015] This application generally relates to novel IL-10 variant molecules that modulate IL-10 receptor signaling. Therefore, this application relates to IL-10 variant molecules in which modifications are incorporated into the IL-10 molecular structure to produce novel IL-10 variant molecules with altered IL-10 receptor binding affinity and / or altered interdomain angles of IL-10. The inventors have surprisingly discovered that modifying IL-10 in key domains affecting IL-10 receptor affinity and / or interdomain angles of IL-10 results in the production of IL-10 receptor agonists, which can be used to treat immune diseases, inflammatory diseases or conditions, and cancer. Furthermore, IL-10 variant molecules can also have an increased serum half-life by incorporating variant IL-10 molecules as part of a fusion protein (e.g., various antibody domains (Fc or variable domains)) without preventing the formation of IL-10 homodimers from IL-10 monomers or variants.

[0016] In some embodiments, the IL-10 variant molecule is modified human IL-10. In other embodiments, the IL-10 variant molecule is modified mouse IL-10. In a preferred embodiment, the IL-10 variant molecule is a modified IL-10 viral homolog. In a more preferred embodiment, the viral IL-10 homolog is CMV-IL10. In the most preferred embodiment, the viral IL-10 homolog is EBV-IL10.

[0017] In a further embodiment, the IL-10 variant molecule incorporates at least one or more amino acid additions, deletions, or substitutions within the receptor-binding domain. In other embodiments, the IL-10 variant molecule incorporates at least one or more amino acid additions, deletions, or substitutions in the region responsible for forming the interdomain angles of IL-10. In a preferred embodiment, the IL-10 variant molecule includes one or both of the modifications in the receptor-binding domain and / or the modifications in the region responsible for the interdomain angles. In the most preferred embodiment, the IL-10 variant molecule incorporates one or both modifications into the EBV-IL10 protein molecule.

[0018] 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 decreased 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 narrower or more restricted interdomain angle compared to wild-type IL-10, and more preferably compared to EBV-IL10. In yet another embodiment, the IL-10 variant molecule forms a wider or more relaxed interdomain angle compared to wild-type IL-10, and more preferably compared to EBV-IL10.

[0019] In some 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 (“site 1”) located in the helix A, AB loop, and / or helix F of EBV IL-10. In some embodiments, modifications to the receptor-binding domain increase or enhance affinity for the IL-10 receptor or receptor complex. In some other embodiments, modifications to the receptor-binding region decrease or reduce affinity for the IL-10 receptor or receptor complex.

[0020] In a further embodiment, the EBV IL-10 variant molecule incorporates at least one or more amino acid additions, deletions, or substitutions within the EBV IL-10 region responsible for interdomain angle formation. In a preferred embodiment, the modification may occur within the DE ring of EBV IL-10. Modifications within the DE ring result in an EBV IL-10 variant molecule having either restricted or relaxed interdomain angles.

[0021] In other respects, IL-10 variant molecules are chimeric or fusion molecules. In one embodiment, the chimeric or fusion protein contains one or more domains derived from different proteins, or contains mutations within a single protein that produce characteristics of another protein. In a preferred embodiment, the chimeric or fusion protein comprises a first portion comprising an IL-10 variant molecule as described herein fused to another molecule, said other molecule including, but not limited to, albumin, enzymes, glycosyltransferases, galactosyltransferases, IgG hinge regions (e.g., Fc regions), one or more variable domains of one or more antibodies (e.g., but not limited to, variable heavy or light chains), cytokines (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, G-CSF, interferon-α, -β, -γ, TGF-β, and tumor necrosis factor-α, -β), markers, pharmaceuticals, chemotherapeutic agents, radioisotopes, and half-life extenders (e.g., hydroxyethyl starch, polysialic acid, heparosan polymers, elastin-like peptides, and hyaluronic acid). In 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 that covalently connect different parts of the fusion protein. A fusion protein can form a non-covalently bound complex with another fusion protein of the same type. Such fusion proteins allow monomers of IL-10 or variant IL-10 molecules to bind together to form a functional homodimer of IL-10 or variant IL-10.

[0022] 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 at a first end of the fusion protein to a linker or spacer, wherein one or more spacers are used to connect the various portions of the fusion protein, and then conjugated to at least one other molecule conjugated at the other end, said molecule being selected from at least one cytokine or its monomer, a therapeutic agent, a marker, a serum half-life prolonging molecule, or a protein (e.g., but not limited to 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 chain and / or light chain variable region via a linker or spacer. In the 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 chain and / or light chain region via a linker or spacer. In the most preferred embodiment, a monomer of EBV IL-10 or a variant thereof is conjugated to a heavy chain variable region and a light chain variable region, wherein the monomers together form a dimer complex. The monomer of EBV IL-10 or a variant thereof may be conjugated to the amino or carboxyl terminus of the heavy or light chain variable region.

[0023] In some embodiments, a therapeutic amount of the present application's IL-10 variant molecule, its fusion protein, or chimeric protein 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, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD). In another embodiment, a therapeutic amount of the present application's IL-10 variant molecule, its fusion protein, or chimeric protein is administered to a subject suffering from cancer. Treatment of subjects suffering from more than one pathological condition is also envisioned. In a more preferred embodiment, the IL-10 variant molecule is an EBV-IL10 variant, its fusion protein, or chimeric protein. In yet another embodiment, the IL-10 variant molecule, its fusion protein, or chimeric protein is used for combination therapy. For example, the IL-10 variant molecule, its fusion protein, or chimeric protein may be administered to a subject in combination with other therapies or treatments. In other embodiments, a therapeutic amount of the present application's IL-10 variant molecule, its fusion protein, or chimeric protein is administered to a subject suffering from a lipid-based disease, such as, but not limited to, elevated cholesterol levels.

[0024] In various embodiments, the IL-10 variant molecule or its fusion protein of this application is delivered as an isolated and purified protein. Delivery may be in the form of subcutaneous 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 an IL-10 variant, its fusion protein, or a 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 using genetic medicine techniques known to those skilled in the art.

[0025] In other embodiments, the IL-10 variant molecule may also be administered as part of a combination therapy regimen. In one embodiment, the IL-10 variant molecule may be administered, for example, in combination with a bispecific T-cell conjugate (BITES), an immunotherapy currently available for the treatment of cancer, IBD, Crohn's disease, NAFLD, NASH, and autoimmune diseases.

[0026] 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 control elements for directing the expression of a sequence encoding an IL-10 variant molecule in target cells, wherein the AAV vector can be administered as a plasmid (“naked” DNA) or packaged in AAV particles. In another embodiment, the nucleic acid vector is a vaccinia virus vector. A variety of vaccinia virus vectors derived from different strains can be used to introduce the variant IL-10 molecule of this application, 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), etc.; all of these strains are available from the United States Center for Type Culture Collection (Manassas, VA).

[0027] In another embodiment, any IL-10 variant molecule described herein, including but not limited to PEGylated IL-10 variant molecules, may be delivered to a subject by any method described herein or known in the art.

[0028] Given the disclosure herein, those skilled in the art will readily conceive of these and other embodiments of the subject matter of this application. Attached Figure Description

[0029] Figure 1 This shows a schematic diagram of the banded structure of the monomeric IL-10 molecule. Josephson people, Immunity(Pages 35-46, 15). The parts highlighted in red represent the site I receptor contact interface, and the parts highlighted in green represent the site II receptor contact interface.

[0030] Figure 2 A-2C was obtained by examining IL-1β in donor 1. Figure 2 A) and TNFα Figure 2 B) Cytokine production and its detection by examining IFNγ ( Figure 2 The production of C) stimulates T-cells, and the ability of IL-10 and EBV IL-10 to activate monocytes / macrophages (Mθ) was compared.

[0031] Figure 3 A-3C was detected by examining IL-1β in donor 1. Figure 3 A) and TNFα Figure 3 B) Cytokine production and its detection by examining IFNγ ( Figure 3 The production of C) stimulates T-cells, and the ability of IL-10 and EBV IL-10 to activate monocytes / macrophages (Mθ) was compared.

[0032] Figure 4 A-4B shows the amount of IFNγ induced from T cells after stimulation with IL-10 or EBV IL-10.

[0033] Figure 5 A-5C shows the effect of the N-terminus of 5 kDa PEGylated and PEGylated EBV-IL-10 on MC / 9 cell proliferation (Fig. 5A), monocyte / macrophage (Mθ) response to LPS TNFα secretion ( Figure 5 B) and T cell response to T cell receptor stimulation of IFNγ secretion ( Figure 5 C).

[0034] Figure 6 A-6E shows the specific amino acid sequences of various IL-10 variant molecules and fusion proteins containing EBV-IL-10.

[0035] Figure 7 A schematic diagram of a fusion protein is shown, comprising IL-10 or an IL-10 variant molecule conjugated to a linker or spacer (in this case, scFv). This representative example shows IL-10 or an IL-10 variant molecule for N-terminal and C-terminal conjugation.

[0036] Figure 8 A-8C is a schematic diagram of various IL-10 variants, such as those prepared in EBV IL-10. Figure 8A(DV05) is an IL-10 variant that contains a single point mutation (V31L V31L) at amino acid position 31 of SEQ ID No. 3. Figure 8 B(DV06) is an IL-10 variant that contains a single point mutation (A75I A75I) at amino acid position 75 of SEQ ID No. 3. Figure 8 C(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.

[0037] Figure 8 D analyzed the responses 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 DV0) inhibited the TNFα secretion response in response to LPS.

[0038] Figure 8 E analyzed the T cell response to various forms of IL-10—wild-type human IL-10, EBV IL-10, DV05, DV06, and DV07—not all forms induce IFN-γ.

[0039] Figure 9 A-9F is a schematic diagram of various configurations of the IL-10 fusion protein / immunoconjugate / dual antibody construct. Figure 9 (a)-(c) represent fusion protein complexes (i.e., biantibodies), wherein each fusion protein contains VH and VL regions obtained from two different antibodies, which are linked to IL-10 monomers (which may also be replaced by IL-10 variant molecules) via a carboxyl-terminal linker or an amino-terminal linker, wherein (a) a single mutation—for example, amino acid position 31—affects IL-10 receptor binding; (b) a single mutation—for example, amino acid position 75—affects IL-10 receptor binding; and (c) two mutations—for example, amino acid positions 31 and 75—affect IL-10 receptor binding. Figures 9(d)-(f) represent fusion protein complexes (i.e., microantibodies), where each fusion protein contains a single VH and VL region obtained from an antibody, which is linked to an IL-10 monomer (which may also be replaced by an IL-10 variant molecule) via a carboxyl-terminal linker or an amino-terminal linker. In (d), a single mutation—for example, amino acid position 31—affects IL-10 receptor binding; in (e), a single mutation—for example, amino acid position 75—affects IL-10 receptor binding; and in (f), two mutations—for example, amino acid positions 31 and 75—affect IL-10 receptor binding.

[0040] Figure 10A-10F is a schematic diagram of various configurations of the IL-10 fusion protein / immunoconjugate / dual antibody construct. Figure 10 (a)-(c) represent single fusion proteins (i.e., mini-antibodies) in which monomers of IL-10 (which may also be replaced by IL-10 variant molecules) are each linked to a VH or VL from the same antibody via a C-terminal or N-terminal linker, and the VH and VL are linked together. The monomers of IL-10 contain (a) a single mutation—e.g., amino acid position 31—affecting IL-10 receptor binding; (b) a single mutation—e.g., amino acid position 75—affecting IL-10 receptor binding; and (c) two mutations—e.g., amino acid positions 31 and 75—affecting IL-10 receptor binding. Figure 10 (d)-(f) represent a single fusion protein in which IL-10 monomers (which may also be replaced by IL-10 variant molecules) are linked together, and each IL-10 monomer is further linked to a single VH or VL region obtained from an antibody via a C-terminal linker or an N-terminal linker. IL-10 monomers contain (d) a single mutation—e.g., amino acid position 31—affecting IL-10 receptor binding; (e) a single mutation—e.g., amino acid position 75—affecting IL-10 receptor binding; and (f) two mutations—e.g., amino acid positions 31 and 75—affecting IL-10 receptor binding.

[0041] Figure 11 This study demonstrates the reduction of tumor volume in vivo using a diabody containing the DV07 mutation. On day 3, a single dose of formulation buffer (1X phosphate-buffered saline; "Control") and the DV07 diabody (a fusion protein complex containing a mature EBV IL-10 variant (with V31L and A75I) and variable domains from anti-CD3α and anti-EGFR, neither of which bind to mouse targets) were administered. For the DV07 diabody, dose concentrations of 1 mg / kg and 0.4 mg / kg were tested.

[0042] Figure 12 The image shows alternative forms of a biantibody against published IL-10 variants (diamonds), wild-type IL-10 (circles), and a biantibody called DHivDEbo:DV07 (diamonds; fusion protein complex containing an EBV IL-10 variant with V31L and A75I mutations and variable domains from anti-HIV and anti-Ebola virus (none of these VH / VL pairs bind to mouse proteins), where the biantibody has Figure 9 (c) Schematic representation of the structure)) in vitro T cell response.

[0043] Figure 13A compared the inhibition of TNFα induced by monocyte / macrophage exposure using different biantibody forms of EBV IL-10 variants with V31L and A75I mutations. “wt” represents human IL-10; “EBV” is EBV IL-10; “DCd3DEgfr:DV05” is a biantibody containing VH and VL regions 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 biantibody containing VH and VL regions from an anti-CD3 antibody and an anti-EGFR antibody linked to an EBV IL-10 variant containing the V31L and A75I mutations; “DHivDEbo:DV07” is a biantibody containing VH and VL regions from an anti-HIV antibody and an anti-Ebola antibody linked to an EBV IL-10 variant containing the V31L and A75I mutations.

[0044] Figure 13 B compared the secretion of IFNγ in T cells using human IL-10 (“wt”) EBV IL-10 (“EBV”) with various biantibody forms containing EBV IL-10 variant molecules with V31L and A75I mutations and VH and VL regions from different antibodies in a reaction assay. The DHivDEgfr:DV07 form is an EBV IL-10 variant biantibody with V31L and A75I substitutions, containing variable regions from anti-HIV and anti-EGFR. The DHivDEbo:DV07 form is an EBV IL-10 variant biantibody with V31L and A75I mutations, containing variable regions from anti-HIV and anti-Ebola.

[0045] Figure 14 A-14B in monocytes / macrophages isolated from two donors ( Figure 14 A) and T cells ( Figure 14 B) compares two forms of EBV IL-10 variant biantibodies: DH:DV07 and DHDE:DV07. The DHDV07 form is an EBV IL-10 variant biantibody with V31L and A75I substitutions, containing variable regions from anti-HIV and anti-EGFR. The DHDE:DV07 form is an EBV IL-10 variant biantibody with V31L and A75I substitutions, containing variable regions from anti-HIV and anti-Ebola. Figure 14 A compared LPS-induced TNFα inhibition in isolated monocytes / macrophages using human IL-10 (“wt”), DH:DV07, and DHDE:DV07. Figure 14B compared the secretion of IFNγ in isolated T cells in response to human IL-10 (“wt”), EBV IL-10, DH:DV07, and DHDE:DV07.

[0046] Figure 15 Various forms of EBV-10 variant biantibody were directly compared on MC / 9 mast cells. This assay compared human IL-10, EBV IL-10, D:DV05 (EBV IL-10 with a V31L mutation containing variable regions from anti-CD3α and anti-EGFR), D:DV06 (EBV IL-10 with an A75I mutation containing variable regions from anti-CD3α and anti-EGFR), D:DV07 with V31L and A75I mutations containing variable regions from anti-CD3α and anti-EGFR), and DhivDEbo:DV07 (EBV IL-10 variant biantibody with V31L and A75I substitutions containing variable regions from anti-HIV and anti-Ebola).

[0047] Figure 16 A-16C is the result of an in vivo tumor study using D:DV07 (with V31L and A75I mutations, containing variable regions from anti-CD3α and anti-EGFR). In vivo tumor volume was assessed after administration of dosing formulation buffer (“Control”), 0.4 mg / kg three times weekly (q3w), 0.2 mg / kg three times weekly (q3w), 0.2 mg / kg every two days (qd), and 0.1 mg / kg every two days (qd).

[0048] Figure 17 A-17B represents in vivo findings from studies of two IL-10 variant fusion protein forms, one high molecular weight and one low molecular weight, corresponding to... Figure 9 Schematic diagrams of C and 9F. IL-10 variants contain V31L and A75I mutations. These results tested the effect of non-targeting IL-10 fusion proteins on reducing tumor size. The VH and VL regions of the fusion protein are non-targeting sequences derived from anti-HIV and anti-Ebola (large) or anti-Ebola (small). Figure 17 A is a non-targeted small-form dosing study in which the drug was administered for 5 days, with a 2-day interval, compared with PEGylated IL-10 (0.75 mg / kg daily). Figure 17 B is a dosing study in non-targeted small (1 mg / kg, 0.5 mg / kg, 0.25 mg / kg) and large (0.2 mg / kg) doses, administered three times weekly, compared with pegylated recombinant human IL-10 (0.75 mg / kg daily).

[0049] Figure 18A-18C represents in vivo research findings on two IL-10 variant fusion protein forms, large and small, respectively corresponding to... Figure 9 Schematic diagrams of C and 9F. These results tested the effect of targeting-capable IL-10 fusion proteins on tumor size reduction. IL-10 variants contain V31L and A75I mutations. In the large form, one set of VH and VL regions of the fusion protein originates from an anti-EGFR antibody, while another set of VH and VL regions originates from an anti-Ebola antibody. The small form includes VH and VL regions derived solely from an anti-EGFR antibody. Figure 18 A represents the results of a study comparing daily administration of the large form (0.25 mg / kg), small form (0.25 mg / kg), and non-targeted small form (0.25 mg / kg) of the IL-10 fusion protein with pegylated recombinant human IL-10 (0.75 mg / kg). Figure 18 B represents the results of a study comparing large-form targeted IL-10 fusion protein (1 mg / kg, 0.25 mg / kg, and 0.25 mg / kg daily) administered three times weekly with large-form non-targeted IL-10 fusion protein (DhDe: DV07, 0.2 mg / kg) and PEGylated IL-10 (0.75 mg / kg daily). Figure 18 C represents the results of a study comparing small-form targeted IL-10 fusion protein (1 mg / kg, 0.25 mg / kg) administered three times weekly with small-form non-targeted IL-10 fusion protein (Debo:DV07, 1 mg / kg) and PEGylated IL-10 (0.75 mg / kg daily).

[0050] Figure 19 A-19B is the result of an in vivo cholesterol study using a biantibody with an EBV IL-10 variant containing a V31L mutation and variable regions derived from anti-CD3α and anti-EGFR.

[0051] Figure 20 A-20B presents the results of an in vitro comparative study of two IL-10 variant fusion proteins on macrophages and T cells. This assay examined the in vitro efficacy of the two forms of the DV06 fusion protein compared to human IL-10. Figure 20 A is a monocyte / macrophage assay that uses DhivDebo:DV06 (SEQ ID No: 26 and 27) and DmadcamDEbo:DV06 (SEQ ID No: 41 and 42). Figure 20 B is a T-cell response assay measured by IFNγ, wherein DhivDebo:DV06 (SEQ ID No: 26 and 27) and DmadcamDEbo:DV06 (SEQ ID No: 41 and 42) are used.

[0052] Figure 21 A-21D is the amino acid sequence of EBV IL-10. Figure 21 A is EBV IL-10. Figure 21 B is DV05 that includes V31L as a substitute. Figure 21 C is DV06, which includes the replacement of A75I. Figure 21 D is the DV07, which includes replacements for V31L and A75I.

[0053] Detailed description of various preferred implementation schemes

[0054] Before describing the application of various embodiments in detail, it should be understood that the application is not limited to specific formulations or process parameters and therefore can, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing various embodiments only and is not intended to be limiting.

[0055] Although a variety of methods and materials similar to or equivalent to those described herein may be used in practice with respect to the various embodiments described herein, preferred materials and methods are described herein.

[0056] Unless otherwise stated, the embodiments described herein employ conventional methods and techniques of molecular biology, biochemistry, pharmacology, chemistry, and immunology well known to those skilled in the art. Many common techniques for designing and manufacturing IL-10 variants, including but not limited to human, CMV, and / or EBV forms of IL-10, and assays for testing IL-10 variants, are well-known methods and are readily available and described in detail in the art. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual* (2nd edition, 1989); *Methods in Enzymology* (edited by S. Colowick and N. Kaplan, Academic Press, Inc.); *Handbook of Experimental Immunology*, Volumes I-IV (edited by DM Weir and CC Blackwell, Blackwell Scientific Publications); AL Lehninger, *Biochemistry* (Worth Publishers, Inc., current addition). Polyglycolization chemistry based on N-terminal aldehydes is also well known in the art.

[0057] The following terms will be used to describe the various embodiments discussed herein and are intended to be defined as follows.

[0058] As used herein in describing the various implementations, the singular forms a(a), an(an), and the(the) include plural references unless the context clearly indicates otherwise.

[0059] The term "approximately" refers to a deviation of 0.0001-5% from a specified number or range. In one embodiment, the term "approximately" refers to a deviation of 1-10% from a specified number or range. In another embodiment, the term "approximately" refers to a deviation of up to 25% from a specified number or range. In a more specific embodiment, the term "approximately" refers to a difference of 1-25% in nucleotide sequence homology or amino acid sequence homology compared to a wild-type sequence.

[0060] The terms “interleukin-10” or “IL-10” refer to a protein comprising two subunits non-covalently linked to form a homodimer, wherein IL-10 is an insert dimer of two six-helix bundles (helical AFs). As used herein, unless otherwise stated, “interleukin-10” and “IL-10” may refer to human IL-10 (“hIL-10”; Genbank accession number NP_000563; or U.S. Patent No. 6,217,857) protein (SEQ ID No: 1) or nucleic acid (SEQ ID No: 2); mouse IL-10 (“mIL-10”; Genbank accession number: 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 may 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 its 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 its nucleic acid (SEQ ID No:6). As used herein, the term monomeric IL-10 refers to the subunits of IL-10 or variant IL-10 that form a homodimer of IL-10 or variant IL-10 when non-covalently linked. The terms “wild-type,” “wt,” and “natural” are used interchangeably herein to refer to the protein sequence (e.g., IL-10, CMV-IL10, or EBV-IL10) of the source species of the particular IL-10 discussed, which is typically found in nature. For example, the terms “wild-type” or “natural” EBV-IL10 therefore correspond to the amino acid sequence most commonly found in nature.

[0061] The terms “derived,” “origin,” “derived from,” or “from” are used herein to identify the original source of a molecule, such as the viral form of the IL-10 molecule, but do not imply limitation on methods of preparing, manufacturing, producing, or producing the molecule. This will include, for example, but not limited to, chemical or recombinant methods.

[0062] The term “derivative” is intended to include any suitable modification of the reference molecule of interest or its analogues, such as sulfation, acetylation, glycosylation, phosphorylation, polymer conjugation (e.g. with polyethylene glycol), hesylation or the addition of other exogenous moieties, as long as the desired biological activity of the reference molecule or variant is preserved (e.g., anti-inflammatory and / or T-cell non-stimulatory).

[0063] The terms “variant,” “analyte,” and “mutant protein” refer to bioactive derivatives of a reference molecule that retain the desired activity, such as anti-inflammatory activity. Generally, when referring to peptides, the terms “variant,” “alternative,” “analyte,” and “mutant protein” refer to one or more compounds having the native peptide sequence and structure but with one or more amino acid additions, substitutions (which are inherently conserved), and / or deletions relative to the native molecule. Therefore, the terms “IL-10 variant,” “variant IL-10,” “IL-10 variant molecule,” and their grammatical variations and plural forms are intended to refer to equivalent terms for IL-10 amino acid (or nucleic acid) sequences that differ from wild-type IL-10 by 1–25% in sequence identity or homology. Thus, for example, an EBV IL-10 variant molecule is a molecule that differs from wild-type EBV IL-10 in that it has one or more amino acid (or nucleotide sequence encoding that amino acid) additions, substitutions, and / or deletions. Therefore, in one form, the EBV IL-10 variant is a molecule that differs from the wild-type sequence of SEQ ID No.:3 by approximately 1% to 25% sequence homology, equivalent to approximately 1–42 amino acid differences.

[0064] The term "fusion protein" refers to a combination or conjugation of two or more proteins or peptides that produces a novel protein arrangement that is not typically naturally occurring. Fusion proteins are formed by the covalent linkage of two or more proteins or peptides. The two or more proteins constituting a fusion protein can be arranged in any conformation from the amino terminus to the carboxyl terminus. Thus, for example, the carboxyl terminus of one protein can be covalently linked to either the carboxyl terminus or the amino terminus of another protein. Exemplary fusion proteins may include the combination of monomeric IL-10 or monomeric variant IL-10 molecules with one or more antibody variable domains. Fusion proteins can also form dimers or bind to other fusion proteins of the same type, thereby forming fusion protein complexes. The complexation of fusion proteins activates or enhances the function of the fusion protein in certain situations 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 a limited or reduced ability to bind to the IL-10 receptor; however, when the fusion protein is complexed, the monomeric form of IL-10 or variant IL-10 molecules becomes a homodimer, and the variable domains bind to form a functional biantibody.

[0065] "Functional variants" are IL-10 variant molecules that include modifications (e.g., additions, substitutions, and / or deletions) that do not impair the biological activity of the reference molecule. These variants may be "homological" to the reference molecule as defined below. Generally, the amino acid sequence of such analogs will have a high sequence homology with the reference sequence, for example, when the two sequences are aligned, the amino acid sequence homology is greater than 50%, typically greater than 60%-70%, and even more specifically greater than 80%-85% or more, such as at least 90%-95% or more. Typically, the analog will include the same number of amino acids but will include substitutions. Functional variants will retain enhanced, diminished, or substantially the same biological activity compared to the native molecule. Specifically, the term "variant" IL-10 molecule is interchangeable with the terms "engineered" IL-10 molecule or IL-10 variant molecule or IL-10 variant, referring to an IL-10 molecule or protein that includes one or two modifications to the IL-10 receptor-binding domain and / or the region responsible for forming inter-domain angles or inter-homogeneous angles in the IL-10 molecule or protein. The terms "variant IL-10 fusion protein," "biantibody," or "fusion" generally refer to the formation of a fusion protein (or fusion protein complex) that comprises variant IL-10 (in monomeric or homodimeric form) and at least one other protein. As used herein, "variant IL-10 or its fusion protein" is used throughout this specification to describe such variant IL-10 fusion proteins.

[0066] "Analogous" can include inherently conservative substitutions. For example, conservative substitutions can include similar substitutions, such as, but not limited to, (1) an acidic substitution between aspartic acid and glutamic acid; (2) a basic substitution between any of lysine, arginine, or histidine; (3) a nonpolar substitution between any of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; and (4) an uncharged polar substitution between any of glycine, asparagine, glutamine, cysteine, serine, threonine, or tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Leucine can also be replaced by isoleucine or valine, aspartic acid by glutamic acid, and threonine by serine, respectively; or similar amino acid-like conservative substitutions can be made with structurally related amino acids, provided that the desired specific biological activity is intact. For example, a polypeptide of interest may include up to about 1-10 conserved or non-conserved amino acid substitutions, or even up to about 15-25 conserved or non-conserved amino acid substitutions, or any integer between 1 and 50, as long as the desired function of the molecule remains intact. Those skilled in the art can readily identify regions of the molecule of interest known in the art that are resistant to variation.

[0067] "Mutant proteins" also include polypeptides having one or more amino acid-like molecules, including but not limited to: compounds containing only amino and / or imino molecules, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids), polypeptides with substituted bonds, and other modifications known in the art, including naturally occurring and non-natural (e.g., synthetic), cyclized, branched molecules, etc. Preferably, the analog or mutant protein will retain enhanced, diminished, or substantially the same biological activity compared to the natural molecule. Methods for preparing polypeptide analogs and mutant proteins are well known in the art.

[0068] The terms “homology,” “homology,” “homologous,” or “substantially homologous” refer to the percentage of identity between at least two polynucleotide sequences or at least two polypeptide sequences. Sequences are homologous to each other when they exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80%-85%, more preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined molecular length.

[0069] The term "sequence identity" refers to the precise correspondence between nucleotides or amino acids. Sequence identity can range from 100% to 50%. Various methods can be used to determine percentage sequence identity, including but not limited to directly comparing the sequence information between two molecules (a reference sequence and a sequence with an unknown percentage of identity compared to the reference sequence) by aligning sequences, counting the exact matches between two aligned sequences, dividing by the length of a reference sequence, and then multiplying the result by 100. Easily available computer programs can be used to help determine percentage identity.

[0070] The term "fragment" is intended to encompass a portion of a molecule that includes a full-length amino acid or polynucleotide sequence and / or structure. Fragments of peptides may include, for example, C-terminal deletions, N-terminal deletions, and / or internal deletions of native peptides. Active or functional fragments of a particular protein typically comprise at least about 5-10 consecutive amino acid residues of the full-length molecule, preferably at least about 15-25 consecutive amino acid residues, most preferably at least about 20-50 or more consecutive amino acid residues, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains biological activity, such as anti-inflammatory activity. When referring to antibodies, an antibody fragment is a portion of a complete antibody containing the antigen-binding site or variable region (heavy chain and / or light chain region) of the complete antibody. These antibody fragments may include, for example, Fab, Fab', Fab'-SH, (Fab')2, Fv fragments, biantibodies, single-chain Fv (ScFv), single-chain peptides having one light chain variable domain, or fragments with three CDRs having either a light chain variable domain or a heavy chain variable domain.

[0071] The term "substantially purified" generally refers to the isolation of a substance such that it constitutes a majority percentage of the sample. The substantially purified component comprises 50% of the sample, preferably 80%-85%, more preferably 90%-95%. Similarly, when referring to polypeptides or polynucleotides, the term "isolated" means that the molecule in question is separate and discrete from the entire organism in which it is found in nature, or exists in the absence of other biomolecules of the same type.

[0072] The terms “subject,” “individual,” or “patient” are used interchangeably in this document and refer to vertebrates, preferably mammals. Mammals include, but are not limited to, rats, rodents, apes, humans, farm animals, sporting animals, and certain pets.

[0073] The term "application" includes the route of administration that allows the applied active ingredient to perform its intended function.

[0074] When referring to, for example, the administration of the EBV-IL-10 variant or its fusion protein described herein, "therapeuticly effective amount" means a sufficient amount of the EBV-IL-10 variant or its fusion protein to promote certain biological activities. These biological activities may include, for example, inhibition of myeloid cell function, enhancement of Kupffer cell activity, and / or deficiency of CD8 receptors. + The role of T cells or enhancement of CD8 + T cell activity and the blocking of mast cell upregulation of Fc receptors or prevention of degranulation. Therefore, an "effective dose" will improve or prevent the symptoms or signs of a medical condition. An effective dose also refers to a dose sufficient to allow or facilitate diagnosis.

[0075] The term "treatment" or "curative action" refers to methods for reducing the effects of a disease or condition. Treatment can also refer to methods for reducing the root cause of the disease or condition itself, not just its symptoms. Treatment can be any reduction in the natural level and can be, but is not limited to, the complete disappearance of the disease, condition, or its symptoms.

[0076] Chemotherapy agents are compounds that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, indomethacin, and piperazine; aziridines such as benzodopa, carboquinone, metodopa, and urodopa; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylene melamine, triethylene ethylphosphamide, triethylene thiophosphamide, and tris(hydroxymethyl)melamine; and nitrogen mustards such as chiorambucil, naphthiamethoxam, cholophosphamide, estradiol, ifosfamide, mechlorethamine, methoxymethacin hydrochloride, melphalan, novobichin, and benzyl mustard. Cholesterol, prednisone, trolophosphamide, uracil mustard; nitrosoureas, such as carmustine, chlorhexidine, formustine, lomustine, nimustine, ramustine; antibiotics, such as aclacinomysin, actinomycin, autramycin, diazoserine, bleomycin, actinomycin C, carrichomycin, carabicin, caminomycin, carcinomycin, chromomycin, actinomycin D, donomycin, detoxin, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin, deoxydoxamycin, idarubicin, ephedrine, mitomycin, mycophenolic acid, norfloxacin. Glycyrrhizin, oliquimycin, pepromycin, potfiromycin, puromycin, triamcinolone acetonide, rhodopsin, streptomycin, streptozotocin, tuberculin, ubenimex, neocarcinomacin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, methotrexate, pteroxate, trimethoprim; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs, such as cyclocytidine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, fluorouridine, 5-FU; androgens, such as capetoresistor, drotaldone propionate, cyclothionin... Alcohols, meandranone, testosterone; anti-adrenergics, such as aminoglutethimide, mitotane, and trilosterone; folic acid supplements, such as folic acid; glucuronolactone; aldehyde phosphoramide glycoside; aminolevulinic acid; acridine; bestrabucil; bifenthrin; edatraxate; defofamine; colchicine; diazinon; elformithine; elifonitrile; etogliflozin; gallium nitrate; hydroxyurea; lentinan; chlordamine; mitotane; mitotane; mopiperol; nitropropionic acid; pentostatin; phenamet; pirarubicin; podophyllinic acid;2-Ethylhydrazide; Procarbazine; PSK®; Razosen; Cizonan; Germanium spiroamine; Alternaria alternifolia ketoacid; Triaminoquinone; 2,2',2”-Trichlorotriethylamine; Urethane; Vinpocetine; Dacarbazine; Mannitol mustard; Dibromomannitol; Dibromoeutherol; Piperobromide; Guasitosine; Ara-C; Cyclophosphamide; Thiotepa; Taxanes, such as paclitaxel (TAXOL® Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere™, Rhone-Poulenc Rorer, Antony, France); Chlorobutyrate; Gemcitabine; 6-Thioguanine; Misopurine; Methotrexate; Platinum analogs, such as cisplatin and carboplatin; Vincristine sulfate; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Noviben; Norfloxacin; Teniposide; Donomycin; Aminopterin; Xeloda® Roche, Switzerland; Ibandronate sodium; CPT11; Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes anti-hormonal agents used to modulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trivoxifen, keoxifene, LY117018, onanasone, and toremifene; and anti-androgen agents, such as flutamide, nilumid, bicalutamide, leuprorelin, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0077] As used in this application, the terms "conjugate," "conjugated," "conjugated," or "conjugated" refer to two or more parts of a molecule linked together. The linking occurs via a covalent bond (e.g., a peptide bond).

[0078] IL-10 variant protein

[0079] The IL-10 variant molecules of this application include modifications to IL-10 in any form. These modifications to the IL-10 molecule include the addition, deletion, and / or substitution of one or more amino acids in the regions and / or domains involved in IL-10 receptor binding and / or in those regions and / or domains that impart angles between domains or between homodimers in the IL-10 molecule. Exemplary IL-10 sequences that can be used to construct the variant IL-10 molecules of this application include, but are not limited to, those 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., Virol. (2000) 268:272-280; Kotenko et al., Proc. Natl. Acad. Sci. USA (2000) 97:1695-1700), equine herpesvirus (see, e.g., Rode et al., Virus Genes (1993) 7:111-116), and OrF virus (see, e.g., Imlach et al., J. Exp. Med. (1995) 182:477-486), cytomegalovirus ... Homologous to Gen. Virol. (2002) 83:1049-1058 and Fleming et al., Virus Genes (2000) 21:85-95. Other representative IL-10 sequences include NCBI accession numbers NM010548, AF307012, M37897, M84340 (mouse sequences); U38200 (horse); U39569, AF060520 (cat sequences); U00799 (cattle); U11421, Z29362 (sheep sequences); L26031, L26029 (macaque sequences); The sequences described in AF294758 (monkey); U33843 (dog); AF088887, AF068058 (rabbit sequences); AF012909, AF120030 (marmot sequences); AF026277 (opossum); AF097510 (guinea pig); U11767 (deer); L37781 (gerbil); and AB107649 (lamb and camel) are as follows.

[0080] 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 mouse (SEQ ID No:7) wild-type protein. Representative examples of various IL-10 variant molecules are provided in SEQ ID Nos. 9-23.

[0081] Modifications relative to wild-type IL-10 include the addition, deletion, and / or substitution of one or more amino acids in regions responsible for (i) IL-10 receptor binding and / or (ii) the angle between domains or between homodimers of the IL-10 molecule.

[0082] Variant IL-10 molecule: IL-10 receptor binding region

[0083] The region responsible for receptor binding includes any amino acid portion located within the region directly involved in or responsible for the binding of IL-10 to IL-10 receptor 1 (IL 10R1) and / or IL-10 receptor 2 (IL 10R2). These regions may include, for example, discontinuous portions of the IL-10 molecule previously discussed and depicted in the art (see, for example, Yoon 2005; Josephson 2001). For example, modifications to any region are contemplated in this application, such as, but not limited to, helical A, helical F, and AB loops responsible for forming the contact points and clefts associated with IL-10 binding to the IL-10 receptor. In a preferred embodiment, modifications to the receptor-binding domain (e.g., addition, deletion, and / or substitution) include amino acids 31 and / or 75 of SEQ ID No. 3. In a particularly preferred embodiment, the modification includes replacing valine at position 31 of SEQ ID No. 3 with leucine (V31L, referred to herein as “DV05”), replacing alanine at position 75 of SEQ ID No. 3 with isoleucine (A75I, referred to herein as “DV06”), or including the substitutions of 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.

[0084] In one implementation, modifications to the receptor-binding domain include those by Josephson et al. (Immunity, 2001, 15, pp. 35-46). Figure 1This refers to any one or more site Ia and / or site Ib interface contacts discussed. In one embodiment of this application, site Ia interface contact includes one or more amino acids located at the bend of helix F and in the AB loop. In another embodiment, site Ib interface contact includes 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 responsible for receptor binding on IL-10 include any one or more of 1-10 amino acids within helix A and 1-7 amino acids within the AB loop. In another embodiment, the receptor binding region may include one or more of the following amino acids or 1-10 amino acids centered thereon, wherein the amino acid is 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.

[0085] In one respect, modifications to the receptor-binding domain include those by Josephson et al. (Immunity, 2001, 15, pp. 35-46). Figure 1 The discussion refers to any one or more site IIa and / or site IIb interface contacts. In one embodiment of this application, the site IIa interface contact comprises one or more amino acids located within the DE ring. In another embodiment, the receptor-binding region may comprise one or more modifications of 1-10 amino acids centered thereon, wherein 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 will contain 1-100 (or any integer thereof) amino acid additions, deletions, and / or substitutions that affect the receptor-binding domain, wherein such additions, deletions, and / or substitutions increase or decrease the binding affinity of the variant IL-10 molecule to the IL-10 receptor.

[0086] Variant IL-10 molecule: Modification of interdomain angles

[0087] The region responsible for forming the interdomain (or homodimer) angles of the IL-10 molecule includes any amino acid portion located within the region directly involved in or responsible for forming the specific interdomain angles of the IL-10 homodimer. Wild-type IL-10 forms an “L-shaped” dimer when the two monomeric units of IL-10 intertwine in an antiparallel manner. The interdomain angles of the resulting human IL-10 and EBV-IL10 have been reported to be approximately 89 degrees and 97 degrees, respectively. To modulate the signaling of the IL-10 receptor, in one embodiment, this application seeks to modify the amino acids responsible for forming the L-shaped dimer within the DE ring, helix D, or helix E of each monomer, which are responsible for forming the interdomain angles. In another embodiment, the region responsible for the interdomain angles includes a linker region of approximately 12 amino acids located between helix D and helix E of the IL-10 protein. Modifications by addition, deletion, or substitution, compared to human IL-10 or EBV-IL10, result in a restricted or relaxed interdomain angle of IL-10. When the monomeric IL-10 molecule is modified to cause homodimerization, resulting in restricted / tight / closed or relaxed / loose / open interdomain angles of the IL-10 domains, the modified IL-10 molecule will produce a variant IL-10 molecule with altered interdomain angles, which binds to and modulates its homologous receptor (IL-10 receptor). In another embodiment, substitution includes introducing proline into an amino acid segment located between the D and E helices and / or between the C and D helices of EBV-IL10.

[0088] Therefore, in one embodiment, when compared to wild-type IL-10, the variant IL-10 molecule will contain one or more additions, deletions, and / or substitutions that exhibit altered intermolecular angles or altered interdomain angles. The altered intermolecular angles or altered interdomain angles can dimerize with the same or different variant IL-10 molecules to produce variant IL-10 molecules that, when compared to wild-type IL-10 molecules, bind to the IL-10 receptor at different binding angles. The different binding angles of the variant IL-10 molecules result in the ability to modulate or “tune” IL-10 receptor signaling to activate or inhibit inflammatory 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 EBV-IL10 molecules as the basis for modification. In yet another embodiment, the variant IL-10 molecule is a hybrid molecule that acquires portions and domains from other IL-10 molecules (e.g., but not limited to human IL-10, mouse IL-10, and / or CMV-IL10).

[0089] In another preferred embodiment, the variant IL-10 molecule produces a relaxed interdomain angle, which will inhibit inflammatory cell (myeloid) responses and does not drive the activation of lymphocytes (such as T cells). When combined with modifications to the receptor-binding domain, preferably with modifications that result in reduced or unchanged receptor affinity, the variant IL-10 molecule with a relaxed interdomain angle will effectively inhibit cytokine secretion by myeloid cells (monocytes, macrophages, neutrophils, granulocytes, mast cells, Kupffer cells) in response to pro-inflammatory stimuli. This conformation of the variant IL-10 molecule can be used, for example, to treat inflammatory diseases such as, but not limited to, IBD, Crohn's disease, psoriasis, rheumatoid arthritis, NAFLD, and NASH.

[0090] In another preferred embodiment, the variant IL-10 molecule produces a restricted interdomain angle, which will enhance the activation of immune cells such as T cells. When combined with modifications to the receptor-binding domain, preferably when combined with modifications that result in higher receptor affinity, the variant IL-10 molecule with a restricted interdomain angle will effectively enhance, for example, CD8+. + T cells, NK cells, and Kupffer cells clear the virus. This conformation of the variant IL-10 molecule can be used, for example, to treat a variety of solid tumors and hematologic malignancies, including metastatic cancers.

[0091] The region responsible for forming the interdomain angle can be a continuous or discontinuous portion within the IL-10 molecule. In one embodiment, the interdomain angle of the IL-10 variant molecule will have a degree variation of 1-25 degrees; in another preferred embodiment, the degree variation is 1-10 degrees; in a more preferred embodiment, the degree variation is 1-5 degrees; and in the most preferred embodiment, the degree variation is less than 5 degrees. In one embodiment, the variant IL-10 molecule will contain 1-100 (or any integer number thereof) amino acid additions, deletions, and / or substitutions that affect the interdomain angle.

[0092] In one embodiment of this application, variant IL-10 molecules are designed and created by predicting one or more regions most responsible for IL-10 receptor binding and / or inter-domain angles, aided by computer modeling. Computer-based modeling will help provide a faster and more efficient method to predict these regions, and modifying them will be most beneficial to the receptor-binding domain and / or inter-domain angles.

[0093] In another embodiment, the molecules of this application comprise derivatives of variant IL-10 molecules. These may include modifications to the variant molecules to include entities that increase the size, half-life, and bioavailability of the variant molecules.

[0094] Variant IL-10 molecule: PEG modified

[0095] In one embodiment, the variant IL-10 molecule may include the addition of polyethylene glycol (PEG). The PEGylated IL-10 variant includes the attachment of at least one PEG molecule. Without being bound by any particular theory, the attachment of PEG to the IL-10 variant can prevent protein hydrolysis, reduce immunogenicity, promote the instability of the IL-10 variant on the receptor to maintain its inhibitory effect on myeloid cells, and prevent T cell activation.

[0096] In its most common form, PEG is a linear or branched polyether terminated by a hydroxyl group, which has a general structure: HO—(CH2CH2O) n —CH2CH2—OH The method of conjugating PEG with the variant IL-10 molecule of this application follows those techniques / methods already established in the art. For example, conjugating or coupling PEG requires activating PEG by preparing a derivative of PEG with functional groups at one or both ends. The most common route for PEG-conjugated proteins is to activate PEG with functional groups suitable for reacting with lysine and N-terminal amino acid groups. In particular, the most common reactive group involved in PEG-peptide conjugation is the α or ε amino group of lysine.

[0097] The reaction of the PEGylation linker with the variant IL-10 molecule results in the PEG moiety being linked primarily at the following sites: the α-amino group at the N-terminus of the protein, the ε-amino group on the lysine residue side chain, and the imidazole group on the histidine residue side chain. In some embodiments, because the variant IL-10 molecule is a recombinant protein with a single α-amino group and multiple ε-amino and imidazole groups, numerous positional isomers can be generated depending on the linker chemistry.

[0098] Two widely used first-generation activated monomethoxy PEGs (mPEGs) are succinimide carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotechnol. Appl. Biochem 15:100-114; and Mironand Wilcheck (1993) Bioconjug. Chem. 4:568-569) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence, et al., U.S. Patent No. 5,650,234), which preferentially react with lysine residues to form carbamate bonds, but are also known to react with histidine and tyrosine residues. It has been shown that the linkage with histidine residues on IFNα is a hydrolytically unstable imidazole carbamate bond (see, e.g., Lee and McNemar, U.S. Patent No. 5,985,263, which is incorporated herein by reference in its entirety).

[0099] Second-generation PEGylation techniques have been designed to avoid these unstable bonds and the lack of selectivity in residue reactivity. The use of PEG-aldehyde linkers targets a single site on the N-terminus of peptide and / or protein subunits via reductive amination. IL-10 can be PEGylated using different types of linkers and pH to obtain various forms of PEGylated molecules (see, for example, U.S. Patent Nos. 5,252,714, 5,643,575, 5,919,455, 5,932,462, 5,985,263, and 7,052,686, which are incorporated herein by reference in their entirety).

[0100] IL-10 mimic molecule

[0101] In another embodiment, this application includes mimicry molecules that reflect the biological function of variant IL-10 molecules. These mimicry molecules include, but are not limited to, peptides, small molecules, modified hormones, and antibodies, having the same or substantially the same structure and / or function as the variant IL-10 molecule. IL-10 mimicry molecules can form the basis for modification to replicate or reflect variant IL-10 molecules, including those mimicry molecules described in US20080139478, US20120238505, and / or US20150218222, all of which are incorporated herein by reference in their entirety.

[0102] IL-10 hybrid molecules and IL-10 fusion protein

[0103] In another embodiment, this application includes an IL-10 variant molecule as a hybrid molecule, which is composed of portions obtained from human IL-10, EBV-IL10, and / or CMV-IL10. For example, different domains in each of human IL-10, EBV-IL10, and / or CMV-IL10 can be combined to produce a hybrid molecule, such that the combination employs all or part of the receptor-binding domain and / or the domain responsible for the inter-domain angle in IL-10.

[0104] In another 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., SSGGGGS (SEQ ID No.: 30, GGGGSGGGGSGGGGS (SEQ ID No.: 31) or SSGGGGSGGGGSGGGGS (SEQ ID No.: 54)), a constant region of an antibody, scFv, or a biantibody. 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 can preferably be a constant heavy chain (CH) region 1, CH2, or CH3. In a more preferred embodiment, the linker of the spacer is a random amino acid sequence of SEQ ID No: 30 and / or 31. Alternatively, the linker or spacer may also contain at least two interchain disulfide bonds.

[0105] The fusion protein may also contain a monomer of at least one IL-10 or IL-10 variant molecule conjugated to the N-terminus, C-terminus, or both ends of the fusion protein. In another embodiment, the fusion protein containing IL-10 or an IL-10 variant may further contain at least one cytokine conjugated to the end opposite to IL-10 or the variant IL-10, and includes IL-2, IL-7, IL-15, IL-26, IL-27, IL-28, IL-29, IL-10, IL-10 variant molecules, IFN-α, TGF-β, basic-FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or any combination thereof. In some preferred embodiments, the fusion protein comprises, in monomeric form, 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; the fusion protein comprises, in monomeric form, 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; 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, IL-10 variant molecules, IFN-α, TGF-β, basic-FGF, EGF, PDGF, IL-4, IL-11, or IL-13.

[0106] In another embodiment, the fusion protein is prepared as follows: using a single-chain variable fragment (scFv), biantibody, Fab, or any antibody fragment as a base scaffold, to which one or two monomers of IL-10, one or two monomers of IL-10 variant molecules, IL-2, IL-7, IL-15, IL-26, IL-27, IL-28, IL-29, IFN-α, TGF-β, basic-FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or combinations thereof, are conjugated.

[0107] In a particularly preferred embodiment, the fusion protein comprises at least one variable region having a variable heavy chain (VH) and / or a 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 a variant IL-10 monomer linked to at least one variable region of the antibody. In one aspect, the fusion protein is a linearly continuous sequence comprising an IL-10 monomer or an IL-10 monomer variant molecule linked to a VH, linked to a VL, and linked to an IL-10 monomer. The variable region of the antibody may be a variable heavy (VH) chain region, a variable light (VL) chain region, or both. The first fusion protein comprises a protein sequence having a linearly continuous configuration such that the IL-10 monomer or variant IL-10 monomer is conjugated to the C-terminus (VH or VL or both) of the variable region. The second fusion protein may comprise a protein sequence having a linearly continuous configuration such that the IL-10 monomer or variant IL-10 monomer is linked to the N-terminus (VH or VL or both) of the variable region. Representative examples of the above-described first fusion protein may include the following configurations: a) NH2 (Ab1VL) COOH -(Connector)- NH2 (Single IL10) COOH Representative examples of the aforementioned second fusion protein may include the following conformations: b) NH2 (Single IL10) COOH -(Connector)- NH2 (Ab1VH) COOH The first (a) and second (b) fusion proteins are joined together in an antiparallel manner to form a functional protein complex, wherein monomers of IL-10 or variant IL-10 attached to the ends form a functional homodimer, and the variable regions together can form a functional antigen-binding site (“ABS”) (see, for example). Figure 9 (a)-(f)).

[0108] In alternative embodiments, the IL-10 monomer or variant IL-10 monomer may be conjugated to at least two variable regions from the same antibody or from two different antibodies. In this configuration, the at least two variable regions are VH and VL. An example of this configuration includes a first fusion protein in which a linear, continuous protein sequence of the VH region of the first antibody is linked at its C-terminus to the N-terminus of the VL region of the second antibody, and subsequently to the N-terminus of the IL-10 monomer or IL-10 variant molecule monomer. An alternative configuration includes a second fusion protein in which a linear, continuous protein sequence of the monomeric IL-10 or IL-10 variant molecule is linked at its C-terminus to the N-terminus of the VH region of the second antibody, and subsequently to the N-terminus of the VL region of the first antibody. Representative examples of the above-described first fusion protein may include the following configurations: a) NH2 (Ab 1- VH) COOH- -(Connector)- NH2 (Ab2VL) COOH -(Connector)- NH2 (Single IL10) COOH Representative examples of the aforementioned second fusion protein may include the following conformations: b) NH2 (Single IL10) COOH -(Connector)- NH2 (Ab2VH) COOH -(Connector)- NH2 (Ab 1- VL) COOH The first (a) and second (b) fusion proteins are joined together in an antiparallel manner to form a functional protein complex, wherein the terminally linked IL-10 or variant IL-10 monomers form a functional homodimer, and the variable regions together can form ABS (see, for example). Figure 9 (a)–(C)).

[0109] In yet another embodiment, the fusion protein comprises two IL-10 monomers or two variant IL-10 monomers 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 conformation, the VH and VL regions may originate from the same antibody or from at least two different antibodies. In a particular conformation of this fusion protein, a VH or VL region is linked to the amino terminus of a first monomer, which is then linked via its carboxyl terminus to the amino terminus of a second monomer, which is then linked to the amino terminus of a VL or VH. Optionally, additional VH or VL regions may be linked to the amino or carboxyl terminus, wherein the VH or VL regions may originate from the same antibody or different antibodies. Representative examples of the above-described fusion proteins may include the following conformations (see, for example, Figure 10 (d)-(f)): NH2 (Ab 1- VH) COOH- -(Connector)- NH2 (Single IL10) COOH -(Connector)- NH2 (Single IL10) COOH- (Connector) -NH2 (Ab 1- VL) COOH The aforementioned fusion protein can fold in a manner that allows IL-10 monomers to form homodimers and the variable domains (VH and VL) of the antibody to form functional ABS.

[0110] In another embodiment, the fusion protein comprises two IL-10 monomers or monomers of two variant IL-10 located at opposite ends of the fusion protein, and at least one VH and VL region, wherein the VH and VL regions are linked together. In this configuration, the VH and VL regions are fused together and each monomer is individually linked to the VL region or VH region of the first antibody. In this configuration, the IL-10 monomer or monomer of the variant IL-10 is each individually linked to the VH or VL of the first antibody. Representative examples of the above-described fusion proteins may include the following configurations (see, for example, Figure 10 (a)-(c)): a) NH2 (Single IL10) COOH -(Connector) -NH2 (Ab 1- VH) COOH- (Connector) NH2 (Ab 1- VL) COOH- (Connector) -NH2 (Single IL10) COOH b) NH2 (Single IL10) COOH -(Connector) -NH2 (Ab 1- VL) COOH- (Connector) NH2 (Ab 1- VH) COOH- (Connector) - NH2 (Single IL10) COOH The monomer or variant of IL-10 can be linked to a VH or VL sequence via a linker sequence. The linker can be a carboxyl-terminal linker, which connects the carboxyl terminus of the variable chain region (VH or VL) to the amino terminus of the IL-10 monomer or IL-10 variant molecule. Alternatively, the linker can be an amino-terminal linker, thereby connecting the carboxyl terminus of the IL-10 monomer or IL-10 variant molecule to the amino terminus of the variable chain region (VH or VL).

[0111] Therefore, 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, wherein the two variable regions are configured such that a VH region from a first antibody is linked to a VL region from a second antibody, or a VL region from a first antibody is linked to a VH region from a second antibody. The fusion protein of this form may comprise a monomeric IL-10 molecule or a variant thereof, comprising at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor. Amino acid substitutions affecting IL-10 receptor binding may occur in human, CMV, or EBV IL-10. The amino acid substitutions may preferably be in EBV IL-10 and include substitutions at positions 31, 75, or both. Amino acid substitutions may include V31L or A75I substitutions or any one or more of both. In addition to amino acid substitutions affecting IL-10 receptor binding affinity, IL-10 variants may also include modifications affecting interdomain angles. In another embodiment, the fusion protein comprises a configuration selected from: (a) a VH region of a first antibody linked at its carboxyl terminus to the amino terminus of a VL region of a second antibody, subsequently linked to the carboxyl terminus of an IL-10 monomer or a variant thereof; or (b) an IL-10 molecule or a variant thereof linked at its carboxyl terminus to the amino terminus of the VH region of a second antibody, subsequently linked to the amino terminus of the VL region of a first antibody. In a preferred embodiment, these fusion protein configurations comprise sequences of SEQ ID Nos.: 24-28, 29, and 33-53. These fusion protein sequences are capable of forming complexes in which the monomer or variant of IL-10 is capable of forming homodimers. Such complexes may comprise and / or be configured as biantibody complexes.

[0112] In another form, the fusion protein can be formulated as an immunoconjugate comprising a first fusion protein and a second fusion protein, wherein the first fusion protein comprises a heavy chain variable region (VH) of a first antibody at its amino terminus, which is linked to a light chain variable region (VL) of a second antibody, the light chain variable region (VL) of the second antibody being further linked to an IL-10 monomer; the second fusion protein comprises an IL-10 monomer at its amino terminus, which is linked to the VH of the second antibody, the VH of the second antibody being further linked to the VL of the first antibody, wherein the VH and VL of the first and second antibodies are combined to form a biantibody, and the IL-10 monomer forms a functional dimerized IL-10 molecule. In another preferred embodiment, the immunoconjugation complex comprises a first fusion protein and a second fusion protein, the first fusion protein comprising a VH region of a first antibody and a monomeric IL-10 molecule linked to its N-terminus; the second fusion protein comprising an IL-10 monomer linked to a VL region of the first antibody at its N-terminus, wherein the VH region of the first antibody binds to the VL region of the first antibody, thereby allowing the monomeric IL-10 molecule on each peptide chain to form a functional IL-10 dimer. The monomeric or variant IL-10 monomer may include, as described above, amino acid modifications affecting IL-10 receptor binding and / or interdomain angles. In another preferred embodiment, the immunoconjugation complex comprises a first fusion protein and a second fusion protein, the first fusion protein comprising a VH region of a first antibody linked to a monomeric IL-10 molecule at its N-terminus; the second fusion protein comprising an IL-10 monomer linked to a VL region of the first antibody at its N-terminus, wherein the VH region of the first antibody binds to the VL region of the first antibody, thereby allowing the monomeric IL-10 molecule on each peptide chain to form a functional IL-10 dimer. In another embodiment, the immunoconjugate comprises at its amino terminus a monomer of a first IL-10 (or IL-10 variant molecule) monomer linked to a VH region of a first antibody, the VH region of the first antibody linked to a VL region of the first antibody, and the VL region of the first antibody linked to a monomer of a second IL-10 (or IL-10 variant molecule), wherein the two IL-10 monomers are capable of binding together to form a functional IL-10 dimer. The described VH and VL regions are capable of forming antigen-binding sites that specifically target antigens (e.g., receptors, proteins, nucleic acids, etc.). Thus, there are two chains together forming a fusion protein complex: chain 1 and chain 2, which produce a functional IL-10 (or IL-10 variant molecule) homodimer. Representative fusion protein chains (i.e., chain 1 and chain 2) include the following:

[0113] The fusion protein comprises a VH and VL pair derived from at least one antibody. The VH and VL pair function as a scaffold to which monomers of IL-10 or its variants can be attached, enabling homodimerization into a functional IL-10 molecule. Therefore, those skilled in the art will understand that the VH and VL scaffolds used in the fusion protein can be selected based on the appropriate physical properties required for the dimerization of the correct IL-10 or IL-10 variant protein and / or to maintain the targeting ability of 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 by other CDR regions to obtain a specifically targeted fusion protein. It is also envisioned that if the fusion protein is not intended to target any specific antigen, the VH and VL pair can be selected as a scaffold that does not target any specific antigen (or antigens with low abundance in vivo), such as VH and VL pairs derived from anti-HIV and / or anti-Ebola antibodies. The fusion protein may contain 1-4 variable regions. The variable regions may be derived from the same antibody or from at least two different antibodies. The antibody variable chain can be obtained or derived from a variety of antibodies, such as those targeting proteins, cell receptors, and / or tumor-associated antigens. In another embodiment, the variable region is obtained from antibodies targeting antigens associated with various diseases (e.g., cancer) or antigens that are not typically found or are rarely found in the serum of healthy subjects. For example, the variable region may be derived from antibodies against 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, for example, the variable region is obtained from or derived from antibodies against EGFR, anti-MadCam, anti-HIV (Chan et al., J. Virol, 2018, 92(18):e006411-19), anti-ICAM, anti-VCAM, or anti-Ebola (US Publication 2018 / 0180614, incorporated herein by reference in its entirety, particularly the mAbs described in Tables 2, 3, and 4). In another embodiment, the variable region is derived from or obtained from an antibody capable of enriching the concentration of a cytokine (e.g., IL-10) to a specific target region so that IL-10 can elicit its biological effect. Such antibodies may include those that target receptors or antigens overexpressed or upregulated in certain diseased regions, or those that are specifically expressed in certain affected regions.For example, the variable region may be derived from antibodies specific to the following: 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; TGFβTrap; 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 just a few. Monomers of IL-10 (e.g., human, CMV, or EBV) or variant IL-10 molecules (described herein) are conjugated to the amino or carboxyl terminus of the variable region (VH or VL) such that the IL-10 or variant IL-10 molecules can dimerize with each other.

[0114] Fusion proteins or fusion protein complexes may also possess antigen-targeting functionality. Fusion proteins or fusion protein complexes contain VH and VL regions that can bind together to form antigen-binding sites or ABS. In some conformations, IL-10 or IL-10 variant molecules or monomers thereof are covalently linked to the end containing the antigen-binding site. These targeting fusion proteins may contain at least one functional variable region or a pair of VH and VL regions at one end of the fusion protein, such that the fusion protein retains the ability to target antigens and has a functional homodimer of IL-10 or IL-10 variant molecules (see [link to relevant documentation]). Figure 9 (a)-(f) and 10(a)-(f)). The variable region can be further modified (e.g., by adding, subtracting, or substituting) by changing one or more amino acids that reduce the antigenicity of the subject. The VH and VL pairs form a scaffold on which CDR regions obtained from multiple antibodies can be grafted. Such antibody CDR regions include those known and described above. For example, CDR regions from any antibody can be grafted onto the VH and VL pairs, such as those described in SEQ ID No: 37, 44, or 45, or those fusion proteins capable of forming fusion protein complexes, such as those described in SEQ ID No: 46 and 47; 48 and 49; or 50 and 51. The CDR regions in the VH and VL scaffolds described above will include the following number of amino acid sites available for CDR grafting / insertion:

[0115] On the other hand, the aforementioned fusion protein can be represented by one of the following general formulas: 1) IL10-L1 -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 (Form IV); 5) IL10-L 1 -X 1 -L 2 -X 2 (Formula V); 6) X 1 -L 1 -IL10 (Form VI); and 7) IL10-L 1 -X 2 (Equation VII) in "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). In a preferred embodiment, "IL-10" is composed of DV05, DV06 or DV07. More preferably, "IL-10" is composed of SEQ ID No: 55, 57 or 59. L 1 "It is a connector of SEQ ID No: 31 or 54; L 2 "It is the connector of SEQ ID No: 30; “X 1"This is from the VH region of the first antibody, which is specific for the following: 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 "The VL region was obtained from the same antibody as X1; “Y 1 "This is from the VH region of the second antibody, which is specific for the following: 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 "The VL region was obtained from the same antibody as Y1; Where X and Y come from the same or different antibodies; “Z” stands for cytokine, which is 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.

[0116] In one embodiment, the substituents of formulas I-VII above are preferably selected from the following: IL-10 is preferably DV05, DV06, or DV07, more preferably IL-10 is composed of DV05, DV06, or DV07, or most preferably IL-10 is composed 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 one of SEQ ID No: 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 that the tag can remain intact or be 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-described antibodies can be replaced by other complementarity-determining regions (CDRs). 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 VH and VL) can be replaced by the six CDR regions of an anti-EGFR antibody (e.g., cetuximab). Therefore, in a preferred embodiment, the fusion protein is SEQ ID No: 33-34, 52, or 53. In another preferred embodiment, the fusion protein is a fusion protein having a scaffold represented by SEQ ID No: 37, 44, 45, 46-47, 48-49, or 50-51; wherein any of the six CDR regions derived from any antibody can be grafted. In other preferred embodiments, the CDR regions from the VH and VL regions of the anti-Ebola antibody can be transplanted from the CDR regions of anti-MAdCAM, anti-VCAM, or anti-ICAM-1, -2, -3, -4 antibodies, wherein in one preferred embodiment, the CDR regions can be transplanted into the fusion protein of SEQ ID No: 37. The fusion proteins of formulas II and III; IV and V; and VI and VII above are designed to bind together to form a bioactive homodimer of IL-10 (or a variant thereof). The above fusion proteins are designed to be untargeted or targeted based on the selected pairs of VH and VL regions and / or the CDR regions transplanted into the VH and VL. The term "untargeted" is intended to describe the inability to target the VH and VL regions of a specific antigen localized in vivo because the antigen is absent or the antigen-binding site (ABS) has been disabled or modified to eliminate ABS function.

[0117] The aforementioned fusion proteins can be further conjugated with accessory proteins / molecules. As used herein, accessory proteins describe proteins conjugated to the fusion protein or fusion protein complex such that they are attached to the opposite side of the IL-10 monomer or variant IL-10 monomer molecule. The addition of accessory proteins effectively creates multifunctional molecules that combine the functions of IL-10 or IL-10 variant molecules with the functions of accessory proteins. The attachment of accessory proteins (e.g., cytokines IL-2, IL-7, IL-12, IL-15, etc.) can, for example, be attached to the N-terminus of the VH region of a fusion protein comprising a VH and VL scaffold. For example, when used in fusion proteins or fusion protein complexes for the treatment of tumors, accessory proteins include, but are not limited to, IL-10, IL-10 variants, 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 used in fusion proteins or fusion protein complexes for the treatment of inflammatory diseases, accessory proteins include, but are not limited to, TGFβ. When used in the treatment of fusion proteins or fusion protein complexes for autoimmune diseases (e.g., but not limited to fatty liver disease), accessory molecules include, but are not limited to, obticholic acid, aramchol, elafibranor, liraglutide, selonsertib, or simtuzumab. When described using Formula IV above, the accessory proteins defined above can be linked to substituents X1 or Y1 on the N-terminal side of the VH portion of the scaffold.

[0118] The aforementioned fusion protein may also include additional amino acid sequences that facilitate the recovery or purification of the fusion protein during manufacturing. These additional amino acid sequences may include various sequence modifications or affinity tags, such as, but not limited to, protein A, albumin-binding proteins, alkaline phosphatases, FLAG epitopes, galactose-binding proteins, histidine tags, and any other tags known in the art. See, for example, Kimple et al. (Curr. Protoc. Protein Sci., 2013, 73: Unit 9.9, Table 9.91, incorporated in its entirety by reference). 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 remain intact. In another embodiment, the affinity tag is a protein A modification incorporated into the fusion protein (e.g., into the VH region of the fusion protein described herein), such as those described in SEQ ID No: 34 or 44-53. Those skilled in the art will understand that any fusion protein sequence described herein can be modified to incorporate a protein A modification by inserting amino acid point substitutions within the antibody frame region, as described in the art.

[0119] In yet another implementation, the various fusion proteins described above can be used in methods for treating cancer, treating or preventing IBD or Crohn's disease, autoimmune diseases, NAFLD, or NASH.

[0120] IL-10 variant polynucleotide

[0121] This application also includes polynucleotide sequences encoding variant IL-10 molecules and the various fusion proteins and / or immune cytokines described above. Once the critical IL-10 receptor-binding region and / or interdomain angles are located in the variant IL-10 molecule of this application, the DNA modifications necessary to achieve the desired modifications in the amino acid sequence are within the skill set of those skilled in the art. Such modifications will be performed using conventional recombinant DNA techniques and methods. For example, synthetic oligonucleotides can be used to introduce the addition or substitution of specific amino acid sequences into the IL-10 sequence at the nucleic acid (DNA) level using site-directed mutagenesis, methods well known in the art.

[0122] In another implementation, the polynucleotide encoding the variant IL-10 sequence can be prepared using standard molecular biology techniques. For example, the polynucleotide sequence encoding the variant molecule can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells expressing the gene, or by deriving the gene from a vector known to contain the gene. The gene of interest can also be synthesized based on a known sequence, rather than cloned. Molecules with suitable codons can be designed for specific sequences. The complete sequence is then assembled from overlapping oligonucleotides prepared using standard methods to form the complete coding sequence. See, for example, Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et al., J. Biol. Chem. (1984) 259:6311.

[0123] In one embodiment, the IL-10 variant molecule or its fusion protein 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. The nucleic acid molecule encoding DV05, DV06, or DV07 may include insertions, deletions, or substitutions (e.g., degenerate codons) that do not alter the function of the IL-10 variant molecule. Due to the degeneracy of the genetic codon, the 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, and may be 70-99% homologous to the aforementioned sequences, 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, the 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 may also be 70%-99% homologous, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous. This application also envisions nucleotide sequences having 70-99% sequence homology 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, preferably having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology. Furthermore, the nucleotide sequences encoding the IL-10 variants and fusion proteins described herein may also include well-known sequences that facilitate, for example, protein expression, production, or secretion. Such sequences may include, for example, leader sequences, signal peptides, and / or translation initiation sites / sequences (e.g., Kozak concordant sequences). The nucleotide sequences described herein may also include one of multiple restriction enzyme sites that allow insertion into various expression systems / vectors.

[0124] In another embodiment, the polynucleotide is contained in a vector that contains the desired IL-10 sequence; or the polynucleotide is artificially synthesized using oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR). 。See, for example, Sambrook, ibid. In another embodiment, the nucleotide sequence encoding the variant IL-10 molecule is obtained by annealing complementary overlapping synthetic oligonucleotides produced in an automated polynucleotide synthesizer, followed by ligation and amplification of the ligated nucleotide sequence by PCR. See, for example, Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. In addition, oligonucleotide directed synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide directed mutagenesis of pre-existing nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic filling of missing oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033) can be used to provide molecules for use in this thematic approach.

[0125] A variety of suitable expression vectors well known to those skilled in the art can be used for the expression and introduction of variant IL-10 molecules and fusion proteins. These vectors include, for example, 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.

[0126] Vectors containing IL-10 variant molecules may also include other vector components required for vector function. For example, the vector may include signal sequences, tag sequences, protease recognition sequences, selection markers, and other sequence regulatory sequences, such as promoters required for the proper replication and expression of the variant IL-10 molecule. There are no particular restrictions on the specific promoters used in the vector, as long as they can drive the expression of the variant IL-10 molecule in multiple host cell types. Similarly, the type of tag promoter is not limited, as long as the tag sequence makes the purification of the expressed variant IL-10 molecule simpler or easier. These tags may include, for example, 6-histidine, GST, MBP, HAT, HN, S, TF, Trx, Nus, biotin, FLAG, myc, RCFP, GFP, etc. There are no particular restrictions on protease recognition sequences; for example, recognition sequences for factors such as Xa, thrombin, HRV, and 3C proteases can be used. There are no particular restrictions on the selected markers, as long as they can detect transformed rice plant cells; for example, neomycin resistance genes, kanamycin resistance genes, hygromycin resistance genes, etc., can be used.

[0127] The resulting DNA construct carrying the desired IL-10 variant or fusion protein can then be used directly for gene therapy or for the production of recombinant IL-10 variants or fusion proteins. In one embodiment, the variant IL-10 molecule or fusion protein of this 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 performed using plasmid DNA or viral vectors, such as adeno-associated virus vectors, adenovirus vectors, retroviral vectors, etc. In some embodiments, the viral vector of this application is administered as viral particles, and in other embodiments, it is administered as a plasmid (e.g., as “naked” DNA).

[0128] Other methods for delivering nucleotide sequences include those known in the art. These methods include delivery via cell-penetrating peptides, hydrophobic portions, electrostatic complexes, liposomes, ligands, liposome nanoparticles, lipoproteins (preferably HDL or LDL), folic acid-targeting liposomes, antibodies (such as folic acid receptors, transferrin receptors), targeting peptides, or via aptamers of nucleotide sequences encoding IL-10 or IL-10 variant molecules, such as, but not limited to, DNA, RNA, siRNA, mRNA, oligonucleotides, or variants thereof. The nucleotide sequences encoding IL-10 variant molecules can be delivered to the subject by direct injection, infusion, patch, bandage, fog or aerosol, or via film delivery. Nucleotides (or proteins) can be delivered to any region where targeted delivery of the desired cytokine stimulant is desired. These regions include, for example, the lungs, gastrointestinal tract, skin, liver, brain (via intracranial injection), and deep metastatic tumor lesions (via ultrasound-guided injection).

[0129] Testing IL-10 variants

[0130] In one embodiment, novel functions of variant IL-10 molecules or their fusion proteins are screened, functions that have not been generated by previous use of IL-10 homodimer sequences, which inhibit macrophage secretion of inflammatory cytokines but do not activate T cells. In one preferred embodiment, the variant IL-10 molecules or their fusion proteins are based on the EBV-IL10 scaffold, which has an anti-inflammatory response but lacks the ability to stimulate T cells. These variant EBV-IL10 molecules or their fusion proteins include modifications to the receptor-binding domain and previously unexplored linker regions, which alter primary, secondary, and tertiary structures to restrict or widen the angles between IL-10 homodimers. In another embodiment, IL-10 variant molecules containing modifications to linker regions or regions responsible for interdomain angle formation have enhanced CD8+ T cell function. In yet another embodiment, IL-10 variant molecules or their fusion proteins containing modifications to linker regions or regions responsible for interdomain angle formation have suppressive bone marrow function but enhanced Kupffer cell function.

[0131] Once a variant IL-10 molecule or its fusion protein is constructed and expressed, those skilled in the art will be able to perform screening assays on the IL-10 variant molecule or its fusion protein to determine whether these molecules possess the desired biological functionality conferred by modifications to the angles between the IL-10 receptor-binding region and / or domains. Various screening assays are known to those skilled in the art and can be used to test for the desired biological function. In one embodiment, the desired biological function includes, but is not limited to, reducing anti-inflammatory responses, reducing T cell stimulation, enhancing T cell function, enhancing Kupffer cell function, and reducing mast cell degranulation.

[0132] For example, IL-10 exposure is known to induce T cells to produce and secrete more IFNγ upon T cell receptor stimulation. Simultaneously, IL-10 exposure prevents monocytes / macrophages from secreting TNFα, IL-6, and other pro-inflammatory cytokines in response to LPS. IL-10 also inhibits FoxP3. + CD4 + T reg Proliferation. In one embodiment, IL-10 variants or their fusion proteins that maximize monocyte / macrophage suppression but lack T cell effector activity (including stimulatory and inhibitory responses) are actively selected. In one embodiment, IL-10 variants or their fusion proteins with increased anti-inflammatory activity are screened for active selection to treat autoimmune diseases, anti-inflammatory diseases, or both. In another embodiment, selection will also be made to enhance Kupffer cell clearance and reduce T cell proliferation. regInhibitory IL-10 variants or their fusion proteins are used to develop treatments for non-alcoholic steatohepatitis (NASH) and / or non-alcoholic fatty liver disease (NAFLD). In yet another embodiment, IL-10 variants that maximize T cell biological function (including stimulatory and inhibitory responses) and also have enhanced Kupffer cell clearance are selected for development into cancer treatments.

[0133] The literature contains numerous descriptions of the effects of measuring cytokines on immune system cells, such as T cells, monocytes / macrophages, Kupffer cells, and T cells. reg Cells and mast cells. This application will apply these assay systems, employing similar assays, to test biological responses by contacting the variant IL-10 molecule or its fusion protein as described in this application.

[0134] Various methods for determining the effectiveness of T-cell responses are described in the prior art. Any of these methods is suitable for testing the variant IL-10 molecule described herein. For example, Chan et al. (2015) described one such method suitable for variant IL-10 molecules. CD8 was isolated from peripheral blood mononuclear cells (PBMCs) using anti-CD8 microbeads. + T cells. Isolated CD8 cells were activated using anti-CD3 and anti-CD28 antibodies. + T cells. Activation can occur, for example, using plates coated with at least approximately 5 to 20 μg / mL anti-CD3 antibody and at least approximately 1 to 5 μg / mL anti-CD28 antibody, over a period of approximately 3 days. After activation, T cells are collected, plated, and treated with a variant of EBV-IL10 or its fusion protein for approximately 3–5 days. Commercially available pegylated recombinant human IL-10 or EBV-IL10 can be used as a control. After treatment with the EBV-IL10 variant, T cells are treated with soluble anti-CD3. Following anti-CD3 treatment, cell culture medium is collected, and interferon-γ (IFNγ) secretion is detected by ELISA.

[0135] Various methods for measuring the stimulation of monocytes / macrophages by cytokines are described in the prior art. Any of these methods is suitable for testing the variant IL-10 molecule described herein. For example, Conway et al. (2017) described one such method suitable for variant IL-10 molecules. Human monocytes were isolated from the erythrocyte sedimentation rate (ESR) amber layer of fresh donor blood using a Ficoll gradient, followed by hypertonic density centrifugation in Percoll. After culturing in RPMI supplemented with 5% human serum and 1% L-glutamine for 30 minutes, the monocytes became adherent and were washed with SMEM Spinner medium to remove contaminated lymphocytes. The test solutions and materials were ensured to be LPS-free. After 4 days of culture, the monocytes / macrophages were contacted or incubated with 10 ng / ml LPS and different concentrations of variant IL-10 molecules for at least 24 hours. The culture supernatant was harvested, and TNF-α and IL-1β concentrations were determined by ELISA.

[0136] Various methods for measuring the response of Kupffer cells to cytokines are described in the prior art. Any of these methods is suitable for testing the variant IL-10 molecule described herein. For example, Chan et al. (2016) described one such method suitable for the variant IL-10 molecule. Kupffer cells were plated in 24-well or 96-well plates and incubated overnight in hepatocyte culture medium (phenol red-free RPMI, pen / strep, Cell Maintenance Supplement B (Invitrogen)). Cells were washed and exposed to the variant IL-10 molecule for 24 hours. Cells were washed once and exposed to 15–20 μl DiI-LDL, DiI-VLDL, DiI-OxLDL, or DiI-AcLDL, 2 μl DMSO, and 15 μl cytochalasin D for 4 hours, after which uptake was measured. All cells were washed once in 1X PBS and lysed with 110 μl cell lysis buffer. 45 μl of cell lysate was transferred to a clear-bottomed, black-walled plate and fluorescence was read at 575 nm.

[0137] Various methods for determining the effectiveness of T-mediated cell responses stimulated with cytokines are described in the prior art. Any of these methods is suitable for testing the variant IL-10 molecule described herein. For example, Chan et al. (2016) described one such method suitable for variant IL-10 molecules. Using CD4... + Microbead separation CD4 +T cells were cultured in AIMV medium for 5–6 days, containing different concentrations of variant IL-10 molecules and 2 μg / mL fixed anti-CD3 and 1 mg / mL anti-CD28. FoxP3 expression in the cells was analyzed by flow cytometry to determine the presence of FoxP3. + CD4 + Whether TGF-β or IL-2 is induced in T regulatory cells.

[0138] Various methods for determining the effectiveness of mast cell proliferation in response to cytokine stimulation are described in the prior art. The mouse mast cell line MC / 9 is a common cell line used to prepare an IL-10 molecule release assay. Specifically, IL-10 and IL-10 variants induce titratable proliferation of mast cells. Conversely, IL-10 inhibits Fc expression in mast cells, indicating that IL-10 has both stimulatory and inhibitory effects on these cells. Any of these methods is suitable for testing the variant IL-10 molecules described herein. For example, Thompson-Snipes et al. (1991) described such a method suitable for variant IL-10 molecules. MC / 9 mast cells were plated in flat-bottomed 24-well plates containing 1 ml RPMI 1640, 10% FCS, 50 mM 2-Me, and different concentrations of variant cytokines. After 3 days of culture, cells were counted using a cell counter to determine the effect of variant IL-10 molecules on mast cell proliferation.

[0139] IL-10 is known to play a role in inhibiting mast cell expression of IgE receptor, FcεRI, and IgE-mediated cytokine production. Therefore, methods for testing the effects of IL-10 on mast cells have been described in the prior art. These methods are suitable for testing the IL-10 variant molecules described herein. For example, Kennedy Norton et al. (2008) described such a method. Human mast cells were isolated from donor skin samples and cultured in a medium containing stem cell factor (SCF) with or without IL-10. FcεRI expression was determined by flow cytometry using an anti-FcεRI specific antibody, followed by FITC-labeled anti-mouse F(ab')2.

[0140] Compositions and formulations containing IL-10 variant molecules

[0141] The IL-10 variant molecule or its fusion protein of this 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 a pharmaceutically acceptable excipient. The pharmaceutical composition may be formulated with commonly used buffers, excipients, preservatives, and stabilizers. The pharmaceutical composition will be formulated for administration to a patient in a therapeutically effective amount sufficient to provide the desired therapeutic outcome. Preferably, such an amount has minimal negative side effects. In one embodiment, the amount of the variant IL-10 molecule or its fusion protein administered will be sufficient to treat an inflammatory disease or condition. In another embodiment, the amount of the variant IL-10 molecule or its fusion protein administered will be sufficient to treat cancer. The dosage may vary from patient to patient and needs to be determined by considering the subject or patient's disease or condition, the patient's overall health status, 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 its fusion protein containing one or both modifications to the receptor-binding domain and / or the angle between the domains of IL-10. In another embodiment, the variant IL-10 molecule is a polyethylene glycolated form of the variant IL-10 molecule. In yet another preferred embodiment, the pharmaceutical composition comprises a variant IL-10 molecule incorporated as a fusion protein or immune cytokine and a pharmaceutical excipient.

[0142] The effective dose for a particular patient can vary depending on factors such as the condition being treated, the patient's overall health, the route and dosage of administration, and the severity of any side effects. The appropriate dose administered to a patient is typically determined by the clinician using parameters or factors known or suspected in the art to affect or expected to affect treatment. Generally, the dose is started slightly below the optimal dose and then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include those related to symptoms such as inflammation or the levels of inflammatory cytokines produced.

[0143] Dosage for patient administration 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, for example, as discussed in detail in U.S. Patents 9,943,568, 10,010,588, and 10,143,726, for example, PEGylated IL-10 is known to improve the circulating half-life of IL-10). Fusion proteins, immunoconjugates, fusion proteins, small antibodies, and biantibodies containing IL-10 or variant IL-10 disclosed herein can also prolong the circulating half-life while maintaining high affinity binding to the IL-10 receptor. Therefore, in one embodiment, various diseases, disorders, or conditions associated with IL-10, or diseases, disorders, or conditions that can be improved by administration of fusion proteins, fusion protein complexes, immunoconjugates, or biantibodies containing IL-10 or variant IL-10, can be administered to patients in need. In a preferred embodiment, diseases, disorders, or conditions associated with IL-10 can be treated or prevented by administering a therapeutically effective amount of an immunoconjugate complex, fusion protein, or biantibody containing EBV IL-10 or variants thereof (including variants affecting IL-10 receptor binding affinity) to a patient in need, wherein the immunoconjugate complex, fusion protein, or biantibody has a molecular weight of about 60 to 155 kDa, and wherein the therapeutically effective amount is in the range of about 0.5 μg / kg to 100 μg / kg. The immunoconjugates, fusion proteins, or biantibodies described herein can be administered daily, three times a week, twice a week, weekly, every two months, or monthly. The EBV IL-10 portion and variable region of the immunoconjugate, fusion protein, or biantibody can be any configuration or combination of those structures discussed herein. Molecules with extended half-lives 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.

[0144] Methods of co-administering or treating with a second therapeutic agent, such as cytokines, steroids, chemotherapeutic agents, antibiotics, anti-inflammatory agents, or radiation, are well known in the art. These may include combination therapy with other therapeutic agents, such as, but not limited to, one or more of the following: interferon-β (e.g., IFNβ-1α and IFN-β-1β); proteins that mimic myelin basic proteins; corticosteroids; IL-1. Inhibitors; TNF inhibitors; anti-TNFα antibodies, anti-IL-6 antibodies, IL-1br-Ig fusion protein, anti-IL-23 antibodies, antibodies against CD40 ligands and CD80; IL-12 and IL-23 antagonists, such as antagonists of the p40 subunits of IL-12 and IL-23 (e.g., inhibitory antibodies against the p40 subunit); IL-22 antagonists; small molecule inhibitors, such as methotrexate, leflunomide, sirolimus (rapamycin) and their analogues, such as 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, their antibodies, such as antibodies against P-selectin); estrogen receptor β (ERB) agonists or ERB-NFkβ antagonists.

[0145] In addition, combination therapy with IL-10 variant molecules or their fusion proteins may include TNF inhibitors, including, for example, chimeric, humanized, effective human, human or in vitro-derived antibodies or antigen-binding fragments thereof that bind to TNF; soluble fragments of TNF receptors, such as p55 or p75 human TNF receptors or their derivatives, such as 75kD TNFR-IgG (75kD TNF receptor-IgG fusion protein, ENBREL™), p55kD TNF receptor-IgG fusion protein; and TNF enzyme antagonists, such as TNFα converting enzyme (TACE) inhibitors. Other combination therapy with anti-inflammatory agents / drugs, including but not limited to standard nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 inhibitors. NSAIDs may include aspirin, celecoxib, diclofenac, diflunisal, etodoxacin, ibuprofen, indomethacin, ketoprofen, ketorolac, nalbometomycin, naproxen, oxaprazin, piroxicam, disalicylate, sulindac, and / or tometetin. The cyclooxygenase-2 inhibitor used in the compositions according to this application may be, for example, celecoxib or rofecoxib.

[0146] Other therapeutic agents that can be co-administered and / or co-formulated with IL-10 variant molecules or their fusion proteins include one or more of the following: interferon-β (e.g., IFNβ-1α and IFNβ-1β); COPAXONE®; corticosteroids; IL-1 inhibitors; TNF antagonists (e.g., soluble fragments of the TNF receptor, such as p55 or p75 human TNF receptors or derivatives thereof, such as 75kdTNFR-IgG; antibodies against CD40 ligands and CD80; and antagonists of IL-12 and / or IL-23, such as antagonists of the p40 subunits of IL-12 and IL-23 (e.g., inhibitory antibodies that bind to the p40 subunits of IL-12 and IL-23); methotrexate, leflunomide, and sirolimus (rapamycin) or analogues thereof, such as CCI-779. Other therapeutic agents may include Imfimzi or Atezolizum.

[0147] For example, for the purpose of treating NASH, IL-10 variants or their fusion proteins can be combined with cholesterol-lowering agents such as statins and non-statins. These agents include, but are not limited to, simvastatin, atorvastatin, rosuvastatin, lovastatin, pravastatin, gemfibrozil, fluvastatin, cholestyramine, fenofibrate, cholesterol absorption inhibitors, bile acid conjugating resins or chelators, and / or microsomal triglyceride transfer protein (MTP) inhibitors.

[0148] An effective dose of the therapeutic agent will affect the level of inflammation or disease or condition by relieving symptoms. For example, the effect may include at least 10%; at least 20%; at least about 30%; at least 40%; at least 50%; or more, resulting in a reduction or complete cure of the disease or condition.

[0149] Pharmaceutical compositions comprising variant IL-10 molecules or their fusion proteins are mixed with pharmaceutically acceptable carriers or excipients. Various pharmaceutical carriers are known in the art and can be used in pharmaceutical compositions. For example, a carrier can be any compatible, non-toxic substance suitable for delivering the variant IL-10 molecule composition of this application to a patient. Examples of suitable carriers include physiological saline, Ringer's solution, dextran solution, and Hank's solution. The carrier may also include any poloxamer commonly known to those skilled in the art, including but not limited to those with 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). The carrier may also include emulsifiers, including but not limited to polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, to name just a few. Non-aqueous carriers, such as non-volatile oils and ethyl oleate, may also be used. The carrier may also include additives, such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives; see, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984). Formulations of therapeutic and diagnostic agents may be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers, for example, in the form of lyophilized powders, slurries, aqueous solutions, or suspensions.

[0150] The applied composition can be administered orally or by injection to the body. Orally administered formulations may further include compounds that protect the variant IL-10 molecule from gastrointestinal proteases. Injection is typically intramuscular, subcutaneous, intradermal, or intravenous. Alternatively, intra-articular injection or other routes may be used where appropriate. Parenteral administration of the variant IL-10 molecule is preferably formulated in a unit-dose injectable form (solution, suspension, emulsion) with a drug carrier and / or pharmaceutically acceptable excipients. In other embodiments, the applied composition may be delivered to the patient via an implantable or injectable drug delivery system.

[0151] Therapeutic uses of IL-10 variants

[0152] In one embodiment, this application provides methods for treating, alleviating, or reducing symptoms associated with inflammation, inflammatory diseases, or autoimmune diseases. This application also provides IL-10, IL-10 variant molecules, fusion proteins, or chimeric molecules thereof as medicaments for inflammation, inflammatory diseases, autoimmune diseases, cancer, or tumors. This application also contemplates the use of IL-10, IL-10 variant molecules, fusion proteins, or chimeric molecules thereof for treating inflammation or inflammatory diseases, or autoimmune diseases, cancer, or tumors. These diseases include, for example, IBD, Crohn's disease, ulcerative colitis, NASH, NAFLD, hypercholesterolemia, or cancer, to name just a few. Methods contemplate administering a therapeutically effective amount of one or more of the variant IL-10 molecules or fusion proteins described herein. In one embodiment, this application includes a method for treating an inflammatory disease or autoimmune disease, the method comprising administering a therapeutically effective amount of a variant IL-10 molecule containing one or more modifications associated with a receptor-binding domain and / or a region responsible for forming the angle between the domains. In a preferred embodiment, the method comprises administering a variant EBV-IL10 molecule or a fusion protein thereof. In a preferred embodiment, the variant IL-10 molecule or its fusion protein for treating inflammatory diseases comprises a variant molecule having restricted interdomain angles and / or also exhibiting lower receptor affinity compared to the wild-type IL-10 molecule. In other embodiments, the variant IL-10 molecule or its fusion protein for treating inflammatory diseases comprises a variant molecule having relaxed interdomain angles and / or also exhibiting lower receptor affinity compared to the wild-type IL-10 molecule. A polyethylene glycol-modified form of the variant IL-10 molecule is also contemplated as part of this application for use in inflammatory diseases or inflammation.

[0153] Inflammatory or autoimmune diseases in this application include any disease or condition associated with unwanted or undesirable inflammatory and immune responses. These diseases include, but are 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, diseases or conditions include neurodegenerative diseases such as Parkinson's disease, amyotrophic lateral sclerosis (ALS), fatal familial insomnia, Rasmussen's encephalitis, Down syndrome, Huntington's disease, Gerstmann-Straussler-Scheinker disease, tuberous sclerosis, neuronal ceroid lipofuscinosis, subacute sclerosing panencephalitis, Lyme disease; tse TSE (African sleeping sickness), HIV dementia, bovine spongiform encephalopathy ("mad cow disease"); Creutzfeldt-Jakob disease; herpes simplex encephalitis, herpes zoster cerebellar inflammation, paralytic dementia (syphilis), tuberculous meningitis, tuberculous encephalitis, optic neuritis, granulomatous vasculitis, temporal arthritis, cerebral vasculitis, Spatz-Lindenberg disease, methamphetamine-associated vasculitis, cocaine-associated vasculitis, traumatic brain injury, stroke, Lance-Adams syndrome, hypoxic-ischemic encephalopathy, radiation necrosis, limbic encephalitis, Alzheimer's disease, progressive supranuclear palsy, striatal substantia nigra degeneration, corticobasal degeneration, primary... Primary progressive aphasia, frontotemporal dementia associated with chromosome 17, spinal muscular atrophy, HIV-associated myelopathy, HTLV-1-associated myelopathy (tropical spastic paraplegia), tabes dorsalis (syphilis), transverse myelitis, post-poliomyelitis syndrome, spinal cord injury, radiation-induced myelopathy, Charcot-Marie-Tooth syndrome, HIV-associated polyneuropathy, Campylobacter-associated motor axonopathy, chronic inflammatory demyelinating polyneuropathy, diabetic muscular avulsion syndrome, phantom limb, complex regional pain syndrome, diabetic neuropathy, paraneoplastic neuropathy, myotonic dystrophy, HTLV-1 Related myopathy, trichinosis, inflammatory myopathy (polymyositis, inclusion body myositis, dermatomyositis), sickle cell disease, α-1-antitrypsin deficiency, tuberculosis, subacute bacterial endocarditis, chronic viral hepatitis, viral cardiomyopathy, Chaga disease, malaria, Coxsackie virus infection, macular degeneration, retinitis pigmentosa, vasculitis, inflammatory bowel disease, rheumatoid arthritis, bullous pemphigus, Churg-Strauss syndrome, myocardial infarction, toxic epidermal necrolysis, shock (such as acute anaphylactic shock), type 1 diabetes, autoimmune thyroiditis, lymphoma, ovarian cancer, lupus (systemic lupus erythematosus), asthma, premature aging, sarcoidosis, type 2 diabetes, and metabolic syndrome.Other inflammation-related diseases or conditions that are embodiments of this application include inflammatory lung diseases such as bronchitis, oxidant-induced lung injury, and chronic obstructive airway disease; inflammatory eye diseases including corneal dystrophy, ocular hypertension, trachoma, onchocerciasis, retinitis, uveitis, sympathetic ophthalmitis, and endophthalmitis; chronic inflammatory gingival diseases including periodontitis; chronic inflammatory joint diseases including arthritis, suppurative arthritis and osteoarthritis, tuberculous arthritis, leprosy arthritis, and tuberous arthritis; skin diseases including sclerosing dermatitis, sunburn, psoriasis, and eczema; encephalomyelitis and viral or autoimmune encephalitis; autoimmune diseases including immune complex vasculitis; and heart diseases including ischemic heart disease, heart failure, and cardiomyopathy. Other non-limiting examples of diseases that may benefit from variant IL-10 molecules or their fusion proteins include: adrenal insufficiency; hypercholesterolemia; atherosclerosis; bone diseases associated with increased bone resorption, such as osteoporosis, preeclampsia, eclampsia, uremia complications; chronic liver failure and other inflammation-related diseases, such as cystic fibrosis, tuberculosis, cachexia, ischemia / reperfusion, hemodialysis-related conditions, glomerulonephritis, restenosis, post-inflammatory sequelae of viral infections, hypoxia, hyperbaric oxygen therapy seizures and poisoning, dementia, Sydenham's chorea, Huntington's disease, epilepsy, Korsakoff's disease, low energy levels associated with cerebrovascular disorders, NO-mediated brain trauma and related sequelae, ischemic cerebral edema (stroke), migraine, vomiting, immune complex disorders, allogeneic transplant rejection, infections caused by invasive microorganisms; and aging.

[0154] The most effective IL-10 variants or fusion proteins thereof for treating anti-inflammatory diseases or conditions include those with reduced T-cell stimulation capacity. Therefore, the inventors of this application have demonstrated that modifying the receptor-binding domain by substitution of the amino acid at position 75 induces minimal T-cell stimulation. In particular, it has been shown that EBV IL-10 with the A75I substitution in SEQ ID No.: 3 (or SEQ ID No. 57) reduces T-cell stimulation (see, for example...). Figure 8 E (represented as DV06). Therefore, a particularly preferred embodiment considers the use of biantibodies and monoantibodies having a variant IL-10 molecule 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 method for treating inflammatory diseases will utilize a fusion protein or fusion protein complex comprising SEQ ID Nos: 26-27; 37; 40; 41-42, 43, 48-49 or combinations thereof.

[0155] In another embodiment of this application, a treatment method includes administering IL-10 or a variant IL-10 molecule or a fusion protein thereof to treat or alleviate cancer-related symptoms. The method considers administering a therapeutically effective amount of one or more of the IL-10 or variant IL-10 molecules or fusion proteins thereof described herein. In one embodiment, this application includes a method of treating or reducing cancer-related symptoms, the method comprising administering a therapeutically effective amount of a variant IL-10 molecule containing one or more modifications associated with a receptor-binding domain and / or a region responsible for forming interdomain angles. In a preferred embodiment, the method comprises administering a variant EBV-IL10 molecule or a fusion protein thereof. Variant IL-10 molecules or fusion proteins thereof for treating cancer include variant molecules having restricted interdomain angles and / or also exhibiting higher receptor affinity compared to wild-type IL-10 molecules. Variant IL-10 molecules or fusion proteins thereof for treating or reducing cancer-related symptoms include variant molecules having relaxed interdomain angles and / or also exhibiting higher receptor affinity compared to wild-type IL-10 molecules. Polyethylene glycolized variants of the IL-10 molecule are also envisioned as part of this application for cancer treatment. A specific example of a fusion protein capable of reducing tumor volume in vivo includes an IL-10 variant with two substitutions at amino acid positions 31 and 75 of SEQ ID No.: 3, which includes specific substitutions V31L and A75I, referred to as DV07 (e.g., SEQ ID No.: 59). Figure 16 AC demonstrated that, at different doses, a fusion protein comprising a DV07 EBV IL-10 molecule conjugated to a biantibody structure, designated D:DV07, reduced tumor volume over 7 and 10 days. Therefore, a particularly preferred embodiment considers the use of biantibodies and monoantibodies having IL-10 variant molecules with a DV07-based mutation (a substitution 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 will utilize a fusion protein or fusion protein complex comprising SEQ ID Nos: 28-29; 33; 34; 35-36; 38-39; 46-47, 61, 63, 65, or 67; or combinations thereof.

[0156] Cancers or proliferative diseases that can be treated with the variant IL-10 molecules or their fusion proteins described herein include a wide range of cancers, including but not limited to uterine cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, penile cancer, gastrointestinal cancers such as esophageal cancer, oropharyngeal cancer, stomach cancer, small or large bowel cancer, colon cancer or rectal cancer, kidney cancer, renal cell carcinoma, bladder cancer, bone cancer, bone marrow cancer, skin cancer, head or neck cancer, liver cancer, gallbladder cancer, heart cancer, lung cancer, pancreatic cancer, salivary gland cancer, adrenal cancer, thyroid cancer, brain cancer such as glioma, ganglion cancer, central nervous system (CNS) and peripheral nervous system (PNS) cancers, and immune system cancers such as spleen or thymus cancer. This application provides methods for treating, for example, immunogenic tumors, non-immunogenic tumors, dormant tumors, and virus-induced cancers, such as epithelial carcinoma, endothelial carcinoma, squamous cell carcinoma, papillomavirus, adenocarcinoma, lymphoma, carcinoma, melanoma, leukemia, myeloma, sarcoma, teratoma, chemically induced cancer, metastasis, and angiogenesis. This application also considers reducing tolerance to tumor cell or cancer cell antigens, for example, by modulating regulatory T cells (T cells). reg ) and / or CD8 + T cell activity. In a preferred embodiment, the IL-10 variant molecule is particularly suitable for treating patients or subjects with liver metastases.

[0157] In other embodiments, methods of treating or reducing symptoms associated with inflammatory diseases or cancer include administering variant IL-10 molecules or their fusion proteins or derivatives thereof (e.g., pegylated) in combination with other therapeutic agents. These therapeutic agents include, but are not limited to, cytokines or cytokine antagonists such as IL-12, IL-2, IL-15, interferon-α or anti-epidermal growth factor receptor, doxorubicin, epirubicin, antifolate, such as methotrexate or fluorouracil, irinotecan, cyclophosphamide, radiation therapy, hormonal or anti-hormonal therapy such as androgens, estrogens, anti-estrogens, flutamide or diethylstilbestrol, surgery, tamoxifen, ifosfamide, dibromoceryl oleracea, alkylating agents such as melphalan or... Cisplatin, etoposide, vinorelbine, vinblastine, vindesine, glucocorticoids, histamine receptor antagonists, angiogenesis inhibitors, radiation, radiosensitizers, anthracyclines, vinblastine alkaloids, taxanes, such as paclitaxel and docetaxel, cell cycle inhibitors, such as cyclin-dependent kinase inhibitors, monoclonal antibodies against another tumor antigen, complexes of monoclonal antibodies and toxins, T-cell adjuvants, bone marrow transplantation or antigen-presenting cells, such as dendritic cell therapy.

[0158] In other embodiments, this application also embodies methods for treating lipid-related disorders (such as hypercholesterolemia and hypertriglyceridemia) and / or improving lipid parameters (such as total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very low-density lipoprotein (VLDL) cholesterol, triglycerides and non-HDL cholesterol), said methods comprising administering a variant IL-10 molecule or a derivative thereof (e.g., PEGylated).

[0159] The most effective IL-10 variants or fusion proteins thereof for treating lipid-related diseases or disorders include those with minimal inhibitory activity against macrophages. Therefore, the inventors of this application have demonstrated that modifying the receptor-binding domain by substituting an amino acid at position 31 (associated with increased inter-domain angles of the homodimer) induces a minimal macrophage response. In particular, it has been shown that EBV IL-10 with the V31L substitution in SEQ ID No.: 3 reduces macrophage responses (see, e.g.) Figure 8 A, denoted as DV05, or SEQ ID No. 55). Therefore, a particularly preferred embodiment considers the use of biantibodies and monoantibodies having an IL-10 variant molecule 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 will utilize a fusion protein or fusion protein complex comprising SEQ ID Nos: 24-25; 50-51; or 45.

[0160] In another embodiment of this application, an IL-10 variant molecule or its fusion protein, viral IL-10 (including EBV or CMV IL-10), or wild-type IL-10 (any of which may optionally include PEGylation or HES-ation) is used in a method of targeting mast cells by reducing mast cell degranulation. In a preferred embodiment, the method of targeting mast cells includes exposure to viral IL-10 or an IL-10 variant molecule to treat seasonal allergic reactions or acute anaphylactic reactions. In another aspect, an IL-10 variant molecule, viral IL-10 (including EBV or CMV IL-10), or wild-type IL-10 is used in a method of reducing IgE reactivity.

[0161] In yet another embodiment, when screening patient populations, the IL-10 variant molecule or its fusion protein of this application is preferably used in the described methods (e.g., anti-inflammatory and / or cancer treatment). In one embodiment, patients exhibiting elevated or high IFNγ response characteristics are most susceptible or ideally suited for cancer treatment using the IL-10 variant molecule. In another embodiment, patients exhibiting decreased or low IFNγ response characteristics are most susceptible or ideally suited for anti-inflammatory treatment using the IL-10 variant molecule.

[0162] The broad scope of this application is best understood by referring to the following embodiments, which are not intended to limit this application to any particular implementation. All citations herein are incorporated by reference, and their scope is specifically and individually mentioned as is the case in each separate publication or patent application.

[0163] Many modifications and variations can be made to this application without departing from the spirit and scope thereof, as will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of example, and this application is limited by the appended claims and the full scope of their equivalents; this application is not limited to the specific embodiments given herein by way of example. Furthermore, all references, patents, and patent applications cited in the foregoing specification are incorporated herein by reference.

[0164] Example

[0165] The following examples are only used to illustrate various implementation schemes of this application and should not be construed as limiting the scope of this application in any way.

[0166] Example 1

[0167] EBV-IL-10 variants or their fusion proteins are constructed by altering the primary sequence using standard molecular biology cloning techniques. Alterations to the primary sequence are designed to change the affinity of the receptor-binding domains and the opening or closing of interdomain angles. Receptor affinity can be altered by changing amino acids at positions 31 and / or 75 and around them in the mature secretory sequence. Interdomain angles can be altered, for example, but not limited to, by introducing proline into the non-α-helical sequence between helices C and D, and between D and E. Proline drives kinking in the linear direction of the primary amino acid sequence, potentially altering the interdomain angles of subsequent domains driven by the secondary and tertiary structures of the D and E helices. Similarly, the introduction of amino acids with large side chains (e.g., tryptophan) can introduce less significant changes to the linear structure of the primary amino acid backbone, resulting in minor alterations to the secondary and tertiary structures.

[0168] Example 2

[0169] The following examples provide a description of how to evaluate variant IL-10 molecules or their fusion proteins in macrophages.

[0170] Human blood was drawn from healthy patients or patients with inflammatory diseases (e.g., Crohn's disease), and the freshly drawn erythrocyte sedimentation rate (ESR) layer was processed using a standard Ficoll density gradient centrifugation procedure to harvest PBMCs. The PBMCs were then subjected to CD14 enrichment using the EasySep™ Human Mononuclear Cell Enrichment Kit (catalog number 19059, Stem Cell Technologies) according to the manufacturer's instructions. + Enrichment of monocytes. Enrichment efficiency was assessed using standard flow cytometry.

[0171] Enriched monocytes at 2 x 10 6 Cells were plated in 24-well plates in RPMI medium supplemented with 5% human serum and PSG. Cells were treated with serial dilutions of variant IL-10 molecules (0, 0.1, 1, 10, 100, 1000 ng / mL) in a 37°C / 5% CO2 humidified incubator for 1 h, followed by exposure to 10 ng / mL LPS (catalog number L4391, Sigma-Aldrich) for 12–16 h. After overnight incubation, the supernatant was collected, and inflammatory cytokines (IL-6, TNFα, IL-1β) were measured by standard ELISA or using the iQue Screener (Intelligentyt).

[0172] In this study, the above method was used to compare the effects of non-PEGylated EBV-IL10 and non-PEGylated human IL-10 on the immunosuppressive capacity of macrophages. Figure 2 A, 2B and 3A, 3B indicate that EBV-IL10 retains its ability to inhibit the inflammatory cytokines IL-1β and TNFα, suggesting that despite differences in the angles between the domains, EBV-IL10 can maintain its anti-inflammatory capacity in a manner similar to human IL-10.

[0173] Example 3

[0174] The following examples demonstrate how to [implement] human CD8 + Description of the evaluation of variant IL-10 molecules or their fusion proteins in T cells.

[0175] Human blood is drawn from healthy patients or patients with inflammatory diseases (e.g., Crohn's disease), and the freshly drawn erythrocyte sedimentation rate (ESR) layer is processed using a standard Ficoll density gradient centrifugation procedure to harvest PBMCs. Then, EasySep™ Human CD8+ is used. +T-cell enrichment kit (catalog number 19053, Stem Cell Technologies) and CD8 assay of PBMCs according to the manufacturer's instructions. + T cell enrichment. Enrichment efficiency was assessed using standard flow cytometry. Enriched cells were suspended in AIMV (Thermo Fisher Scientific, catalog number 12055083) medium. Cells were incubated at 37°C / 5% 002 humidified incubator, washed 1-2 times with 1X PBS, and coated with 10 μg / mL anti-CD3 (catalog number 160039-85, Thermo Fisher Scientific) and 2 μg / mL anti-CD28 (catalog number 160289-85, Thermo Fisher Scientific) for 2 hours.

[0176] Enriched CD8 + T cells (3x10) 6 Add ( / mL / well) to anti-CD3 / anti-CD28 coated plates and incubate at 37°C / 5% CO2 for 72 hours.

[0177] After 72 hours, cells were harvested, counted, and 100 μl were re-coated into 96-well round-bottom plates (2 x 10⁻⁶). 5 Cells / well, with or without serially diluted (0, 0.1, 1, 10, 100, 1000 ng / mL – added at 100 μL / well) variant IL-10 molecules or control samples. Tests were performed in triplicate. Cells containing variant IL-10 molecules or their fusion protein were incubated at 37°C / 5% CO2 for 72 hours. After 72 hours, cells were collected, washed, and re-plated into new round-bottom 96-well plates containing soluble anti-CD3 (catalog number 16-0039-85, Thermo Fisher Scientific), and incubated at 37°C / 5% CO2 for 4 hours.

[0178] In the study, the above method was used to compare the effects of non-PEGylated EBV-IL10 and non-PEGylated human IL-10 on CD8. + T-cell stimulation. Figure 2 C and 3C showed that EBV-IL10 exhibited reduced IFNγ (a measure of T cell stimulation) levels compared to human IL-10. This suggests that T cell stimulation can be modulated by altering the interdomain angles. Figure 4A and 4B showed that half of the treated donors exhibited the expected complete anti-inflammatory effect, while the other half did not. The variant selected for development would mimic the response of donor 1, completely suppressing macrophage secretion of inflammatory cytokines in response to LPS, and lacking activated CD8. + T cell-induced IFNγ. Donor 2 exhibited similar inhibition of monocyte / macrophage secretion of inflammatory cytokines as donor 1, but only shifted the curve of T cell IFNγ secretion and maximum activation to the right. IL-10 variants altering receptor affinity and interdomain angles should further reduce T cell activation in patients similar to donor 2.

[0179] Example 4

[0180] As previously described, human monocytes / macrophages, T cells, and mouse MC / 9 cells purchased from ATCC were cultured, and responses to single or double N-terminal 5 kDa PEGylated EBV-IL10 were evaluated. PEGylation of EBV-IL10 resulted in a slightly reduced macrophage response to LPS. Figure 5 B), but almost completely inhibits IFNγ induction in stimulated T cells ( Figure 5 C). Similarly, PEGylation of EBV-IL10 almost eliminated its stimulatory effect on MC / 9 cells. Figure 5 A).

[0181] Various forms of EBV-IL-10 variant biantibodies containing anti-CD3α and anti-EGFR VH and VL regions were tested using the MC / 9 cell proliferation assay. The EBV-10 variants included D:DV05 (EBV IL-10 with a V31L mutation), D:DV06 (EBV IL-10 with an A75I mutation), and D:DV07 (with both V31L and A75I mutations). Additionally, the DV07 biantibody containing anti-HIV and anti-Ebola VH and VL regions was also tested. The various variant biantibody forms were compared with human IL-10 and EBV IL-10. Results were presented in... Figure 15 Provided by China.

[0182] Other forms of the EBV-IL-10 fusion protein were also tested in vitro. Specifically, DhivDebo:DV06 (SEQ ID No: 26 and 27) and DmadcamDebo:DV06 (SEQ ID No: 41 and 42) were compared with human IL-10 in the macrophage and T cell response assays described herein. Results were... Figure 20 Provided in A and 20B.

[0183] Example 5

[0184] The following examples provide representative protocols for testing IL-10 and IL-10 variants and their fusion proteins in in vivo tumor models. All in vivo studies were conducted in accordance with standard operating procedures and established guidelines approved by the Institutional Animal Care and Use Committee (“IACUC”).

[0185] Purchase eight-week-old female Balb / C mice, isolate them for one week, and maintain them on standard food and water, with bedding changed once a week and a standard 24-hour light / dark cycle.

[0186] CT26 tumor cells (2 x 10) 5 The CT26 tumor was suspended in Hanks' buffered saline solution and subcutaneously implanted into eight-week-old mice to allow for growth. The mice carried CT26 tumors (average 50-150 mm). 3 Wild-type Balb / C (Envigo) or B-cell knockout (Jackson) mice were treated subcutaneously (back of the neck) for 10 days with IL-10 or IL-10 variants or their fusion proteins (e.g., EBV IL-10 variants with two receptor-binding substitutions, DV07 (Fig. 8C), covalently linked to VH and VL from two different antibodies or a biantibody). Tumor length and width were measured every three days using electronic calipers, and tumor volume (L x W) was calculated. 2 B cells in wild-type mice were depleted by intravenous (iv) administration of 200 μg / mouse anti-mouse CD20. Figure 16 The results of such a study are provided, which used an IL-10 variant molecule called D:DV07, which is an IL-10 variant with V31L and A75I mutations and contains variable regions from anti-CD33α and anti-EGFR.

[0187] exist Figure 17 In A and 17B, two forms of the IL-10 variant fusion protein (i.e., [formula missing]) were compared in an in vivo tumor model. Figure 9 C (large form) and 9f (small form) represent IL-10 variants containing V31L and A75I mutations (DV07). The fusion protein is a non-targeted fusion protein containing VH and VL regions from both the anti-HIV and anti-Ebola antibody (large form), and VH and VL regions from the anti-Ebola antibody. Dosing studies examined the effects of a 5-day dosing regimen followed by a 2-day withdrawal of the small form of the non-targeted IL-10 fusion protein compared to pegylated recombinant human IL-10 (0.75 mg / kg daily). Figure 17A). Dosing studies also examined the effects of administration of the large and small forms of the non-targeted IL-10 fusion protein three times weekly compared to pegylated IL-10 (0.75 mg / kg daily). Figure 17 B).

[0188] Studies have also been conducted in vivo using small and large forms of IL-10 fusion proteins with tumor-targeting capabilities (i.e., IL-10 variants containing V31L and A75I mutations, DV07). Figure 18 A represents the results of daily administration of various targeted IL-10 variant fusion proteins, which compares the large form (DegfDebo:DV07) and small form (Degf:DV07) with the small form non-targeted (Debo:DV07) IL-10 fusion protein and PEGylated IL-10. Figure 18 B represents the results of administration of various large-form targeted IL-10 variant fusion proteins three times a week, in which different doses (1 mg / kg and 0.25 mg / kg) of the large form (DegfDebo:DV07) were compared with the small-form non-targeted (Debo:DV07) IL-10 fusion protein and PEGylated IL-10. Figure 18 C represents the results of administration of various small-form targeted IL-10 variant fusion proteins three times a week, in which different doses (1 mg / kg and 0.25 mg / kg) of the small form (Degf:DV07) were compared with the small-form untargeted (Debo:DV07) IL-10 fusion protein and PEGylated IL-10.

[0189] Example 6

[0190] The following examples provide representative protocols for testing IL-10 and IL-10 variants and their fusion proteins in in vivo cholesterol models. All in vivo studies were performed in accordance with standard operating procedures and established guidelines approved by the IACUC.

[0191] Eight-week-old female C57BL / 6J mice were purchased from a suitable supplier, isolated for one week, and kept on standard food and water, with bedding changed weekly and a standard 24-hour light / dark cycle.

[0192] Eight-week-old female C57BL / 6J mice from the Jackson Laboratory were fed a high-fat diet (Envigo) for three weeks. Plasma samples were obtained by post-frame blood collection from each mouse and then treated with IL-10 or a variant of IL-10 or its fusion protein (e.g., an EBV IL-10 variant containing a single substitution (V31L) at amino acid position 31 of SEQ ID No:3, linked as a biantibody (D:DV05 EBV IL-10 variant). Mice were subcutaneously treated with 0.4 and 0.2 mg / kg (3 times weekly (q3w)) and 0.2 and 0.1 mg / kg (weekly (qd), 5 days of treatment, 2 days apart). Animals were treated for two weeks, and blood was collected at the end of the treatment period to quantify plasma cholesterol concentrations before and after administration. One day prior to the start of treatment, 200 μg / mouse anti-mouse CD20 depleted B cells were administered intravenously (iv). Figure 19 The results of such a study are provided in A and 19B.

[0193] Example 7

[0194] The following examples provide representative protocols for testing IL-10 and its variants and fusion proteins in an in vivo sodium dextran sulfate (“DSS”) inflammation model. All in vivo studies were performed in accordance with standard operating procedures and established guidelines approved by the IACUC.

[0195] Eight-week-old female Balb / C mice were purchased from an appropriate supplier, isolated for one week, and maintained on standard food and water, with bedding changed weekly and a standard 24-hour light / dark cycle. B-cell knockout (Jackson) mice were fed water containing 4% DSS for 6 days with free access to food, followed by normal water. On day 5, mice were treated subcutaneously (neck and back) for 10 days with 0.4 and 0.2 mg / kg (3 times weekly (q3w)) and 0.2 and 0.1 mg / kg (daily (qd) for 5 days, with a two-day break). Mice were evaluated daily. 1.) Weight 2.) Blood in stool 3.) Total Blood Volume 4.) Stool consistency Disease activity index is determined by combining scores; 1. Weight loss 2. Stool consistency 3. Bleeding (divided by 3) Each score is determined as follows: weight change (0: <1%, 1: 1-5%, 2: 5-10%, 3: 10-15%, 4: >15%); rectal bleeding (0: negative, 2: positive); or total bleeding (4); and stool consistency (0: normal, 2: loose stool, 4: diarrhea).

[0196] List of preferred implementation schemes

[0197] 1. An Epstein-Barr virus IL-10 (EBV-IL10) variant protein comprising one or more amino acid additions, deletions, and / or substitutions, exhibiting altered interdomain angles and / or altered affinity for a homologous receptor compared to wild-type EBV-IL10, wherein the altered interdomain angles modulate the angle of binding to the homologous receptor upon dimerization.

[0198] 2. The EBV-IL10 protein according to the aforementioned embodiments, wherein one or more amino acid additions, deletions, and / or substitutions are located in the IL-10 receptor binding domain.

[0199] 3. The EBV-IL10 protein according to any of the foregoing embodiments, wherein one or more amino acid additions, deletions and / or substitutions are located within α-helix A and / or helix D.

[0200] 4. The EBV-IL10 protein according to any of the foregoing embodiments, wherein one or more amino acids are added, deleted, and / or substituted in the linker domain of EBV-IL10.

[0201] 5. The EBV-IL10 protein according to any of the foregoing embodiments, wherein one or more amino acid additions, deletions and / or substitutions are located within the DE ring of EBV-IL10.

[0202] 6. The EBV-IL10 protein according to any of the foregoing embodiments, wherein one or more amino acid additions, deletions and / or substitutions are located in a 12-amino acid linker region between α-helix D and α-helix E or α-helix C and α-helix D, preferably the addition or substitution of proline in the 12-amino acid linker region.

[0203] 7. The EBV-IL10 protein according to any of the foregoing embodiments, wherein the affinity for homologous receptor alterations includes the addition, deletion, and / or substitution of one or more amino acids in the IL-10 receptor binding domain.

[0204] 8. The EBV-IL10 protein according to any of the foregoing embodiments further comprises one or more amino acid additions, deletions and / or substitutions located within α-helix A and / or α-helix D.

[0205] 9. The EBV-IL10 protein according to any of the foregoing embodiments further comprises one or more amino acid additions, deletions, and / or substitutions located within the IL-10 receptor binding domain.

[0206] 10. The EBV-IL10 protein according to any of the foregoing embodiments further comprises one or more amino acid additions, deletions and / or substitutions located within α-helix A and / or α-helix D.

[0207] 11. The EBV-IL10 protein according to any of the foregoing embodiments, wherein one or more amino acids are added, deleted and / or substituted at amino acid position 31 and / or position 75 of SEQ ID No. 3.

[0208] 12. A monomeric recombinant protein comprising six α-helices numbered AF capable of forming homodimers with the same monomeric protein, wherein α-helices D and E are linked by interchain amino acid linkers, the linkers being modified by the addition, deletion or substitution of at least one amino acid to alter the intermolecular angles of the protein upon homodimerization.

[0209] 13. The recombinant protein according to the foregoing embodiments, wherein the recombinant protein is a protein derived from a virus.

[0210] 14. The recombinant protein according to any of the foregoing embodiments, wherein the virus is Epstein-Barr virus (EBV).

[0211] 15. The recombinant protein according to any of the foregoing embodiments, wherein the homodimer formed between two identical monomeric proteins forms a specific interaction angle with its homologous receptor.

[0212] 16. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle is greater than that of the natural wild-type protein.

[0213] 17. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle formed during homodimerization results in the protein having a higher affinity for the homologous receptor.

[0214] 18. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle formed during homodimerization results in the protein having a lower affinity for the homologous receptor.

[0215] 19. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle is smaller than that of the natural wild-type protein.

[0216] 20. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle results in the protein having a higher affinity for the homologous receptor.

[0217] 21. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle results in the protein having a lower affinity for the homologous receptor.

[0218] 22. The recombinant protein according to any of the foregoing embodiments, wherein the monomeric protein is interleukin-10.

[0219] 23. The recombinant protein according to any of the foregoing embodiments, wherein the monomeric protein is EBV-IL10.

[0220] 24. The recombinant protein according to any of the foregoing embodiments, wherein the protein angle is conferred by adaptor modification resulting in an angle of interaction with the homologous receptor.

[0221] 25. A recombinant variant of Epstein-Barr virus IL-10 (EBV-IL10) protein comprising at least one amino acid addition, deletion or substitution of the linker region between the D and E helices of EBV-IL10 and / or the receptor-binding region of EBV-IL10.

[0222] 26. The recombinant protein according to the foregoing embodiments, wherein the interaction of the variant EBV-IL10 protein with the same protein results in the homodimer having a modified interaction angle with its homologous receptor, and / or a modified angle between homodimers.

[0223] 27. The recombinant protein according to any of the foregoing embodiments, wherein the variant EBV-IL10 protein forms an interaction angle greater than that of the wild-type EBV-IL10 protein and / or an altered inter-homomer angle.

[0224] 28. The recombinant protein according to any of the foregoing embodiments, wherein the variant EBV-IL10 protein forms an interaction angle smaller than that of the wild-type EBV-IL10 protein and / or an altered angle between homodimers.

[0225] 29. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle formed during homodimer formation results in the variant EBV-IL10 protein having increased affinity for its homologous receptor.

[0226] 30. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle formed during homodimer formation results in the variant EBV-IL10 protein having reduced affinity for its homologous receptor.

[0227] 31. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle confers increased affinity to its homologous receptor.

[0228] 32. The recombinant protein according to any of the foregoing embodiments, wherein the interaction angle confers reduced affinity to its homologous receptor.

[0229] 33. An isolated recombinant polynucleotide encoding a protein according to any of the foregoing embodiments.

[0230] 34. An isolated recombinant polynucleotide encoding a protein according to any of the foregoing embodiments.

[0231] 35. A vector comprising nucleic acid encoding a protein according to any of the foregoing embodiments.

[0232] 36. A host cell comprising a polynucleotide according to any one of the foregoing embodiments.

[0233] 37. A method for treating or preventing inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of a variant protein according to any of the foregoing embodiments.

[0234] 38. The method according to the aforementioned implementation scheme, wherein the angle of change of the variant protein is smaller than that of wild-type EBV-IL10.

[0235] 39. The method according to any of the foregoing embodiments, wherein the variant protein binds to the IL10 receptor with moderate affinity compared to wild-type EBV-IL10.

[0236] 40. The method according to any of the foregoing 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 allergic reaction.

[0237] 41. A method for treating or preventing an autoimmune disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a variant protein according to any of the foregoing embodiments.

[0238] 42. The method according to the aforementioned implementation scheme, wherein the angle of change of the variant protein is smaller than that of wild-type EBV-IL10.

[0239] 43. The method according to any of the foregoing embodiments, wherein the variant protein binds to the IL10 receptor with moderate affinity compared to wild-type EBV-IL10.

[0240] 44. A method for treating or preventing IBD or Crohn's disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a variant protein according to any of the foregoing embodiments.

[0241] 45. The method according to the foregoing embodiments, wherein the angle of change of the variant protein is smaller than that of wild-type EBV-IL10.

[0242] 46. ​​The method according to any of the foregoing embodiments, wherein the variant protein binds to the IL10 receptor with moderate affinity compared to wild-type EBV-IL10.

[0243] 47. A method for treating or preventing nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH) in a subject, the method comprising administering to the subject a therapeutically effective amount of a variant protein according to claim 1.

[0244] 48. The method according to the foregoing implementation scheme, wherein the angle of change of the variant protein is smaller than that of wild-type EBV-IL10.

[0245] 49. The method according to any of the foregoing embodiments, wherein the variant protein binds to the IL10 receptor with moderate affinity compared to wild-type EBV-IL10.

[0246] 50. A method for treating or preventing cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a variant protein according to any of the foregoing embodiments.

[0247] 51. The method according to the aforementioned implementation scheme, wherein the angle of change of the variant protein is greater than that of wild-type EBV-IL10.

[0248] 52. The method according to any of the foregoing embodiments, wherein the variant protein binds to the IL10 receptor with increased affinity compared to wild-type EBV-IL10.

[0249] 53. An engineered fusion protein comprising a monomer of at least one IL-10 or IL-10 variant molecule conjugated to the first end of the fusion protein, at least one cytokine or a monomer thereof conjugated to the second end of the fusion protein, and a linker or spacer. The connector or spacer connects the first and second ends.

[0250] 54. The fusion protein according to the foregoing embodiments, wherein the linker or spacer is a constant region of the antibody.

[0251] 55. The fusion protein according to any of the foregoing embodiments, wherein the constant region is derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD or IgE.

[0252] 56. The fusion protein according to any of the foregoing embodiments, wherein the linker or spacer further comprises at least two interchain disulfide bonds.

[0253] 57. The fusion protein according to any of the foregoing embodiments, wherein the linker or spacer is scFv, a biantibody, or a fragment thereof.

[0254] 58. The fusion protein of any of the foregoing embodiments, wherein the constant region is heavy chain constant (CH) region 1, CH2, CH3 or any combination thereof.

[0255] 59. A fusion protein according to any of the foregoing embodiments, wherein at least one IL-10 or IL-10 variant molecule is conjugated at the N-terminus, C-terminus, or both of the fusion protein.

[0256] 60. The fusion protein according to any of the foregoing embodiments, wherein at least one cytokine conjugated at the other end comprises IL-10, IL-10 variants 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.

[0257] 61. The fusion protein according to any of the foregoing 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.

[0258] 62. A fusion protein according to any of the foregoing 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.

[0259] 63. The fusion protein according to any of the foregoing 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.

[0260] 64. A fusion protein according to any of the foregoing 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.

[0261] 65. A fusion protein v, 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.

[0262] 66. A fusion protein according to any of the foregoing 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.

[0263] 67. The fusion protein according to any of the foregoing embodiments, wherein the C-terminus further comprises IL-10, IL-10 variant molecules, 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.

[0264] 68. A fusion protein according to any of the foregoing embodiments, wherein the fusion protein is prepared on a single-stranded variable fragment (scFv) scaffold.

[0265] 69. The fusion protein according to any of the foregoing embodiments, wherein the fusion protein is prepared on a dual antibody scaffold.

[0266] 70. A fusion protein according to any of the foregoing embodiments, wherein the fusion protein is prepared on a Fab scaffold.

[0267] 71. A fusion protein according to any of the foregoing embodiments, wherein the fusion protein is compounded with another fusion protein, the other 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, wherein the linker or spacer connects the first and second ends.

[0268] 72. A method of treating cancer in a subject in need, the method comprising administering to the subject an engineered fusion protein according to any of the foregoing embodiments.

[0269] 73. A method for treating or preventing IBD or Crohn's disease, the method comprising administering to a subject an engineered fusion protein according to any of the foregoing embodiments.

[0270] 74. A method for treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a subject, the method comprising administering to the subject an engineered fusion protein according to any of the foregoing embodiments.

[0271] 75. A method for activating CD8-positive T cells, the method comprising administering an engineered fusion protein according to any of the foregoing embodiments.

[0272] 76. The method according to any of the foregoing embodiments, wherein the application is in vitro.

[0273] 77. The method according to any of the foregoing embodiments, wherein the administration is administered to a subject in need, wherein the subject has been diagnosed with cancer, IBD or Crohn's disease, or NAFLD or NASH.

[0274] 78. The method according to any of the foregoing embodiments, wherein the fusion protein comprises IL-10 or an 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.

[0275] 79. A method of treating cancer in a subject in need, the method 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 containing IL-10 or an IL-10 variant molecule.

[0276] 80. The method according to the foregoing embodiments, 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 connecting the first and second ends.

[0277] 81. The method according to any of the foregoing embodiments, wherein the IL-10, IL-10 variant molecules, or engineered fusion proteins containing IL-10 or IL-10 variant molecules increase and maintain T cell receptor complex (CD3) signaling.

[0278] 82. A method for treating or preventing inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

[0279] 83. The method according to the foregoing embodiments, wherein the nucleotide sequence is a variant of DNA, RNA or modified thereof.

[0280] 84. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is mRNA or a modified mRNA linked to a nucleoside.

[0281] 85. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is capable of expressing a variant IL-10 molecule in vivo in cells, tissues or organisms.

[0282] 86. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is delivered to a cell, tissue or organism via a cell-penetrating peptide, a hydrophobic portion, an electrostatic complex, a liposome, a ligand, a liposome nanoparticle, a lipoprotein (preferably HDL or LDL), a folic acid-targeting liposome, an antibody (such as a folic acid receptor, transferrin receptor), a targeting peptide, or via an aptamer.

[0283] 87. A method for treating or preventing an autoimmune disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

[0284] 88. The method according to the foregoing embodiments, wherein the nucleotide sequence is a variant of DNA, RNA or a modified version thereof.

[0285] 89. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is mRNA or a modified mRNA linked to a nucleoside.

[0286] 90. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is capable of expressing a variant IL-10 molecule in vivo in cells, tissues or organisms.

[0287] 91. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is delivered to a cell, tissue or organism via a cell-penetrating peptide, a hydrophobic portion, an electrostatic complex, a liposome, a ligand, a liposome nanoparticle, a lipoprotein (preferably HDL or LDL), a folic acid-targeting liposome, an antibody (such as a folic acid receptor, transferrin receptor), a targeting peptide or via an aptamer.

[0288] 92. A method for treating or preventing IBD or Crohn's disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

[0289] 93. The method according to the foregoing embodiments, wherein the nucleotide sequence is a variant of DNA, RNA, or a modified version thereof.

[0290] 94. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is mRNA or a modified mRNA linked to a nucleoside.

[0291] 95. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is capable of expressing a variant IL-10 molecule in vivo in cells, tissues or organisms.

[0292] 96. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is delivered to a cell, tissue or organism via a cell-penetrating peptide, a hydrophobic portion, an electrostatic complex, a liposome, a ligand, a liposome nanoparticle, a lipoprotein (preferably HDL or LDL), a folic acid-targeting liposome, an antibody (such as a folic acid receptor, transferrin receptor), a targeting peptide or via an aptamer.

[0293] 97. A method for treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a subject, the method comprising administering to the subject a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

[0294] 98. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is a variant of DNA, RNA, or a modified version thereof.

[0295] 99. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is mRNA or a modified mRNA linked to a nucleoside.

[0296] 100. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is capable of expressing a variant IL-10 molecule in vivo in cells, tissues or organisms.

[0297] 101. The method according to any of the foregoing embodiments, wherein the nucleotide sequence is delivered to a cell, tissue or organism via a cell-penetrating peptide, a hydrophobic portion, an electrostatic complex, a liposome, a ligand, a liposome nanoparticle, a lipoprotein (preferably HDL or LDL), a folic acid-targeting liposome, an antibody (such as a folic acid receptor, transferrin receptor), a targeting peptide or via an aptamer.

[0298] 102. A fusion protein comprising a monomeric IL-10 molecule or a variant thereof linked to two variable regions from at least two different antibodies, wherein the two variable regions are configured such that a heavy chain variable (VH) region from a first antibody is linked to a light chain variable (VL) region from a second antibody, or a VL region from the first antibody is linked to a VH region from the second antibody.

[0299] 103. The fusion protein according to any of the foregoing embodiments, wherein the monomeric IL-10 molecule or a variant thereof comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

[0300] 104. A fusion protein according to any of the foregoing embodiments, wherein the monomeric IL-10 molecule or a variant thereof comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0301] 105. The fusion protein according to any of the foregoing embodiments, wherein the monomeric IL-10 molecule or a variant thereof is a homolog of Epstein Barr virus (EBV) IL-10 of SEQ ID No.: 3.

[0302] 106. The fusion protein according to any of the foregoing embodiments, wherein the EBV Il-10 homolog includes an amino acid substitution at position 31, position 75, or both.

[0303] 107. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog includes an amino acid substitution at position 31.

[0304] 108. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog includes an amino acid substitution at position 75.

[0305] 109. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog includes amino acid substitutions at positions 31 and 75.

[0306] 110. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog includes a V31L amino acid substitution.

[0307] 111. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog includes an A75I amino acid substitution.

[0308] 112. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises V31L and A75I amino acid substitutions.

[0309] 113. The fusion protein according to any of the foregoing 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.

[0310] 114. The fusion protein according to any of the foregoing 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.

[0311] 115. The fusion protein according to any of the foregoing embodiments, wherein the fusion protein is an amino acid sequence selected from SEQ ID No.: 24-28, 29, 33-51, 61, 63, 65 or 67.

[0312] 116. The fusion protein according to any of the foregoing embodiments, wherein the fusion protein is a biantibody.

[0313] 117. A fusion protein according to any of the foregoing embodiments, wherein the fusion protein comprises a configuration selected from: (a) a VH region of a first antibody linked at its carboxyl terminus to the amino terminus of a VL region of a second antibody, wherein the VL region of the second antibody is subsequently linked to the amino terminus of an IL-10 monomer or a variant thereof; or

[0314] (b) An IL-10 molecule or a variant thereof is attached at its carboxyl terminus to the amino terminus of the VH region of a second antibody, which is then attached to the amino terminus of the VL region of a first antibody.

[0315] 118. A fusion protein according to any of the foregoing embodiments, wherein the configurations (a) and (b) together form a biantibody complex.

[0316] 119. The fusion protein according to any of the foregoing embodiments further comprises a linker located between the VH region and the VL region.

[0317] 120. The fusion protein according to any of the foregoing embodiments, wherein the amino acid sequence is selected from SEQ ID No: 24-28, 29, 33-53, 61, 63, 65, 67.

[0318] 121. The fusion protein of any of the foregoing embodiments, wherein the first antibody and the second antibody comprise one or more amino acid substitutions that reduce antigenicity in the subject.

[0319] 122. An immunoconjugation complex comprising i) a first fusion protein having a heavy chain variable region (VH) of a first antibody at its amino terminus linked to a light chain variable region (VL) of a second antibody, the light chain variable region (VL) of the second antibody being further linked to an IL-10 monomer or a variant thereof; and ii) a second fusion protein having an IL-10 monomer or a variant thereof at its amino terminus linked to a VH of a second antibody, the VH of the second antibody being further linked to a VL of the first antibody, wherein the VH and VL of the first and second antibodies are bound together to form a biantibody, and the IL-10 monomer forms a functional dimerized IL-10 molecule.

[0320] 123. An immunoconjugation complex according to any of the foregoing embodiments, wherein the IL-10 monomer comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

[0321] 124. An immunoconjugate complex according to any of the foregoing embodiments, wherein the IL-10 molecule comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0322] 125. An immunoconjugate complex according to any of the foregoing embodiments, wherein the IL-10 monomer is a homolog of Epstein Barr virus (EBV) IL-10 of SEQ ID No. 3.

[0323] 126. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV Il-10 homolog comprises an amino acid substitution at position 31, position 75, or both.

[0324] 127. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises an amino acid substitution at position 31.

[0325] 128. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises an amino acid substitution at position 75.

[0326] 129. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises amino acid substitutions at positions 31 and 75.

[0327] 130. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises a V31L amino acid substitution.

[0328] 131. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises an A75I amino acid substitution.

[0329] 132. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises V31L and A75I amino acid substitutions.

[0330] 133. An immune conjugate complex according to any of the foregoing 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.

[0331] 134. An immunoconjugate complex according to any of the foregoing embodiments, wherein the first fusion protein is an amino acid sequence selected from SEQ ID No.: 24, 26, 28, 35, 38, 41, 46, 48 or 50.

[0332] 135. An immunoconjugate complex according to any of the foregoing embodiments, wherein the second fusion protein is an amino acid sequence selected from SEQ ID No.: 25, 27, 29, 36, 39, 42, 47, 49 or 51.

[0333] 136. The immune conjugate complex according to any of the foregoing embodiments, further comprising a linker located between the VH region and the VL region.

[0334] 137. An immunoconjugate complex according to any of the foregoing embodiments, wherein the IL-10 monomer in the first fusion protein is linked to the VL region via its amino terminus.

[0335] 138. An immunoconjugate complex according to any of the foregoing embodiments, wherein the IL-10 monomer in the second fusion protein is linked to the VH region via its carboxyl terminus.

[0336] 139. An immunoconjugate complex of any of the foregoing embodiments, wherein the first antibody and the second antibody comprise one or more amino acid substitutions that reduce antigenicity in the subject.

[0337] 140. A biantibody comprising a first peptide chain and a second peptide chain, the 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; the second peptide chain comprising VH and VL from a second antibody, and a monomeric IL-10 molecule, wherein the VH region of the first antibody binds to the VL region of the first antibody, and the VH region of the second antibody binds to the VL region of the second antibody, thereby allowing the monomeric IL-10 molecule on each peptide chain to form a functional IL-10 dimer.

[0338] 141. A biantibody according to any of the foregoing embodiments, wherein the monomeric IL-10 comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

[0339] 142. The biantibody according to any of the foregoing embodiments, wherein the monomeric IL-10 molecule comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0340] 143. The biantibody according to any of the foregoing embodiments, wherein the monomeric IL-10 molecule is a homolog of Epstein Barr virus (EBV) IL-10 of SEQ ID No. 3.

[0341] 144. The biantibody according to any of the foregoing embodiments, wherein the EBV Il-10 homolog comprises an amino acid substitution at position 31, position 75, or both.

[0342] 145. A biantibody according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog contains an amino acid substitution at position 31.

[0343] 146. A biantibody according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog contains an amino acid substitution at position 75.

[0344] 147. A biantibody according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises amino acid substitutions at positions 31 and 75.

[0345] 148. The biantibody according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises a V31L amino acid substitution.

[0346] 149. The biantibody according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises an A75I amino acid substitution.

[0347] 150. The biantibody according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises V31L and A75I amino acid substitutions.

[0348] 151. The biantibody according to any of the foregoing 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.

[0349] 152. The biantibody according to any of the foregoing embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID No.: 24, 26, 28, 35, 38, 41, 46, 48 or 50.

[0350] 153. The biantibody according to any of the foregoing embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID No.: 25, 27, 29, 36, 39, 42, 47, 49, or 51.

[0351] 154. The biantibody according to any of the foregoing embodiments further comprises a linker located between the VH region and the VL region.

[0352] 155. A biantibody according to any of the foregoing embodiments, wherein the first antibody and the second antibody comprise one or more amino acid substitutions that reduce antigenicity in the subject.

[0353] 156. A biantibody according to any of the foregoing embodiments, wherein the IL-10 monomer is linked to the first and second peptide chains via its carboxyl terminus.

[0354] 157. A biantibody according to any of the foregoing embodiments, wherein the first antibody and the second antibody comprise one or more amino acid substitutions that reduce antigenicity in the subject.

[0355] 158. An immunoconjugation complex comprising i) a first fusion protein having a heavy chain variable (VH) region of a first antibody and a monomeric IL-10 molecule linked through its N-terminus; and ii) a second fusion protein having an IL-10 monomer linked to a light chain variable (VL) region of the first antibody at its N-terminus, wherein the VH region of the first antibody binds to the VL region of the first antibody to allow the monomeric IL-10 molecule on each peptide chain to form a functional IL-10 dimer.

[0356] 159. An immunoconjugate complex according to any of the foregoing embodiments, wherein the IL-10 monomer comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

[0357] 160. An immunoconjugate complex according to any of the foregoing embodiments, wherein the IL-10 molecule comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0358] 161. An immunoconjugate complex according to any of the foregoing embodiments, wherein the IL-10 monomer is a homolog of Epstein Barr virus (EBV) IL-10 of SEQ ID No. 3.

[0359] 162. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV Il-10 homolog comprises an amino acid substitution at position 31, position 75, or both.

[0360] 163. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises an amino acid substitution at position 31.

[0361] 164. An immunoconjugated complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises an amino acid substitution at position 75.

[0362] 165. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises amino acid substitutions at positions 31 and 75.

[0363] 166. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises a V31L amino acid substitution.

[0364] 167. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises an A75I amino acid substitution.

[0365] 168. An immunoconjugate complex according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises V31L and A75I amino acid substitutions.

[0366] 169. An immunoconjugate complex according to any of the foregoing embodiments, wherein the VH and VL regions of the first antibody are derived from an anti-epidermal growth factor receptor (EGFR) monoclonal antibody.

[0367] 170. An immunoconjugate complex of any of the foregoing embodiments, wherein the first fusion protein further comprises a VL region from a second antibody that links a VH region of the first antibody to monomeric IL-10, and wherein the second fusion protein further comprises a VH region from a second antibody that links monomeric IL-10 to the VL region.

[0368] 171. An immune conjugate complex according to any of the foregoing embodiments, wherein the VH and VL regions of the second antibody are derived from an anti-Ebola monoclonal antibody.

[0369] 172. An immunoconjugate complex according to any of the foregoing embodiments, wherein the variable region is connected to the IL-10 monomer via a linker.

[0370] 173. An immunoconjugate complex according to any of the foregoing embodiments, wherein the first antibody and the second antibody comprise one or more amino acid substitutions that reduce antigenicity in the subject.

[0371] 174. An immunoconjugate complex according to any of the foregoing embodiments, wherein the first antibody and the second antibody comprise one or more amino acid substitutions that reduce antigenicity in the subject.

[0372] 175. A fusion protein comprising variable light (VL) and variable heavy (VH) regions of a first antibody fused to an IL-10 monomer, wherein the IL-10 monomers are directly linked to each other.

[0373] 176. The fusion protein according to any of the foregoing embodiments, wherein the IL-10 monomer is linked from the carboxyl terminus of the first IL-10 monomer to the amino terminus of the second IL-10 monomer.

[0374] 177. A fusion protein according to any of the foregoing embodiments, wherein the fusion protein comprises, in an amino-to-carboxyl-terminus configuration, the VL region of the first antibody being linked to a first IL-10 monomer, the first IL-10 monomer being linked to a second IL-10 monomer, and the second IL-10 monomer being linked to the VH region of the first antibody.

[0375] 178. The fusion protein according to any of the foregoing embodiments further comprises 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 a second antibody linked to the carboxyl terminus of the VH region of the first antibody.

[0376] 179. The fusion protein according to any of the foregoing embodiments, wherein each of the IL-10 monomers comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

[0377] 180. The fusion protein according to any of the foregoing embodiments, wherein each of the IL-10 monomers comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

[0378] 181. The fusion protein according to any of the foregoing embodiments, wherein the IL-10 monomer is a homolog of Epstein Barr virus (EBV) IL-10 of SEQ ID No.: 3.

[0379] 182. The fusion protein of claim 181, wherein the EBV Il-10 homolog comprises an amino acid substitution at position 31, position 75, or both.

[0380] 183. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises an amino acid substitution at position 31.

[0381] 184. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog contains an amino acid substitution at position 75.

[0382] 185. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises a V31L amino acid substitution.

[0383] 186. The fusion protein according to any of the foregoing embodiments, wherein the EBV-IL-10 homolog comprises an A75I amino acid substitution.

[0384] 187. The fusion protein according to any of the foregoing embodiments, wherein the first antibody is an anti-Ebola monoclonal antibody.

[0385] 188. The fusion protein according to any of the foregoing embodiments, wherein the first antibody is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody.

[0386] 189. The fusion protein according to any of the foregoing embodiments, wherein the first antibody is an anti-Ebola monoclonal antibody and the second antibody is an anti-EGFR monoclonal antibody.

[0387] 190. A method of treating a disease, ailment, or condition in a patient in need, the method comprising administering to the patient a therapeutically effective amount of an Epstein Barr virus (EBV) IL-10 immunoconjugate complex, wherein the molecular weight of the immunoconjugate complex is about 60 to 155 kDa, wherein the therapeutically effective amount is in the range of about 0.5 μg / kg to 100 μg / kg, and wherein the EBV IL-10 portion of the immunoconjugate is derived from SEQ ID No.: 3.

[0388] 191. The method according to any of the foregoing embodiments, wherein the immune conjugate complex is applied monthly, every two months, weekly, twice a week, three times a week, or daily.

[0389] 192. The method according to any of the foregoing 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.

[0390] 193. The method according to any of the foregoing embodiments, wherein the variant EBV IL-10 comprises an amino acid substitution at position 31, position 75, or both of SEQ ID No.: 3.

[0391] 194. The method according to any of the foregoing embodiments, wherein the variant EBV IL-10 comprises a V31L amino acid substitution.

[0392] 195. The method according to any of the foregoing embodiments, wherein EBV IL-10 comprises an A75I amino acid substitution.

[0393] 196. The method according to any of the foregoing embodiments, wherein EBV IL-10 comprises V31L and A75I amino acid substitutions.

[0394] 197. The method according to any of the foregoing embodiments, wherein the immunoconjugate complex is a complex of two fusion proteins.

[0395] 198. The method according to any of the foregoing embodiments, wherein the immunoconjugate complex comprises i. a first fusion protein having a heavy chain variable region (VH) of a first antibody at its amino terminus linked to a light chain variable region (VL) of a second antibody, the light chain variable region (VL) of the second antibody further linked to a carboxyl terminus of an EBV IL-10 monomer; and ii. a second fusion protein having an EBV IL-10 monomer at its amino terminus linked to a VH of a second antibody, the VH of the second antibody further linked to a VL of the first antibody, wherein the VH and VL of the first and second antibodies bind to form a biantibody, and the EBV IL-10 monomer forms a functional dimerized EBV IL-10 molecule.

[0396] 199. The method according to any of the foregoing embodiments, wherein the first antibody and the second antibody are different antibodies.

[0397] 200. The method according to any of the foregoing embodiments, wherein the first antibody is an anti-HIV monoclonal antibody and the second antibody is an anti-Ebola monoclonal antibody.

[0398] 201. The method of claim 202, wherein the fusion protein is an amino acid sequence selected from SEQ ID No: 24-51.

[0399] 202. The method of claim 202, wherein the first fusion protein is an amino acid sequence selected from SEQ ID No: 24, 26, 28, 35, 38, 41, 46, 48 or 50.

[0400] 203. The method of claim 202, wherein the second fusion protein is an amino acid sequence selected from SEQ ID No.: 25, 27, 29, 36, 39, 42, 47, 49 or 51.

[0401] 204. The method according to any of the foregoing embodiments, wherein the immunoconjugate complex is a biantibody comprising an EBV IL-10 monomer fused at any end, wherein the EBV IL-10 monomer is capable of binding to form a functional EBV IL-10 dimer.

[0402] 205. The method according to any of the foregoing embodiments, wherein the disease, patient or condition is selected from cancer, inflammatory disease, autoimmune disease or cholesterol.

[0403] 206. The method according to any of the foregoing embodiments, wherein the EBVIL-10 immunoconjugate complex is administered in an amount sufficient to maintain a stable serum IL-10 concentration, based on administration at least every 2 to 3 days.

[0404] 207. The method according to any of the foregoing embodiments, wherein the immunoconjugate is capable of inhibiting TNFα secretion and inducing IFNγ production at a concentration similar to that of wild-type IL-10.

[0405] 208. The method according to any of the foregoing embodiments, wherein the EBV IL-10 immunoconjugate complex has activity similar to wild-type IL-10.

[0406] 209. The method according to any of the foregoing embodiments, wherein the immunoconjugate complex comprises

[0407] (A) The VH region of the first antibody at the N-terminus is linked to the VL region of the second antibody, which is linked to the carboxyl terminus of the IL-10 molecule; and

[0408] (b) An IL-10 molecule, which is linked to the VH region of a second antibody, which is linked to the VL region of a first antibody.

[0409] 210. A method of treating cancer in a patient in need, the method comprising administering a biantibody to the patient, the biantibody comprising a first peptide chain and a second peptide chain, the first peptide chain having a heavy chain variable region (VH) from the first antibody, a light chain variable region (VL) from the second antibody, and a monomeric IL-10 molecule; the second peptide chain having VH and VL from the second antibody, and a monomeric IL-10 molecule, wherein the VH region of the first antibody binds to the VL region of the first antibody, and the VH region of the second antibody binds to the VL region of the second antibody, thereby allowing the monomeric IL-10 molecule on each peptide chain to form a functional IL-10 dimer.

[0410] 211. The method according to any of the foregoing embodiments, wherein the monomeric IL-10 is a homolog of Epstein Barr virus (EBV) IL-10 of SEQ ID No.: 3.

[0411] 212. The method according to any of the foregoing embodiments, wherein the EBV IL-10 contains an amino acid substitution at position 31 of SEQ ID No.: 3.

[0412] 213. The method according to any of the foregoing embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

[0413] 214. The method according to any of the foregoing 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.

[0414] 215. The method according to any of the foregoing embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID No.: 28, 35, 38, 46.

[0415] 216. The method according to any of the foregoing embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID No.: 29, 36, 39, 47.

[0416] 217. The method according to any of the foregoing embodiments further includes a connector located between the VH region and the VL region.

[0417] 218. The method according to any of the foregoing embodiments, wherein VH and VL each comprise one or more amino acid substitutions that reduce antigenicity in the patient.

[0418] 219. The method according to any of the foregoing 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.

[0419] 220. The method according to any of the foregoing embodiments, wherein the biantibody is formed of two peptide chains having an amino-to-carboxyl-terminal configuration: (a) a first peptide comprising a VH region of a first antibody linked to a VL region of a second antibody, wherein the VL region of the second antibody is then linked to the amino terminus of an IL-10 monomer or a variant thereof; and

[0420] (B) A second peptide containing an IL-10 monomer is linked to the VH region of a second antibody, and then the VH region of the second antibody is linked to the VL region of a first antibody.

[0421] 221. A method of treating cholesterol in a patient in need, the method comprising administering to the patient a biantibody that lowers cholesterol levels, the biantibody comprising a first peptide chain and a second peptide chain, the first peptide chain having a heavy chain variable region (VH) from the first antibody, a light chain variable region (VL) from the second antibody, and a monomeric IL-10; the second peptide chain having VH and VL from the second antibody, and a monomeric IL-10 molecule, wherein the VH region of the first antibody binds to the VL region of the first antibody, and the VH region of the second antibody binds to the VL region of the second antibody, thereby allowing the monomeric IL-10 molecule on each peptide chain to form a functional IL-10 dimer.

[0422] 222. The method according to any of the foregoing embodiments, wherein the monomeric IL-10 is a homolog of Epstein Barr virus (EBV) IL-10 of SEQ ID No.: 3.

[0423] 223. The method according to any of the foregoing embodiments, wherein the EBV IL-10 contains an amino acid substitution at position 31 of SEQ ID No.: 3.

[0424] 224. The method according to any of the foregoing embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

[0425] 225. The method according to any of the foregoing 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.

[0426] 226. The method according to any of the foregoing embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID No.: 24 or 50.

[0427] 227. The method according to any of the foregoing embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID No.: 25 or 51.

[0428] 228. The method according to any of the foregoing embodiments further includes a connector located between the VH region and the VL region.

[0429] 229. The method according to any of the foregoing embodiments, wherein VH and VL each comprise one or more amino acid substitutions that reduce antigenicity in the patient.

[0430] 230. The method according to any of the foregoing 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.

[0431] 231. The method according to any of the foregoing embodiments, wherein the biantibody is formed from two peptide chains having the following amino-to-carboxyl terminal configuration: (a) A first peptide comprising a VH region of a first antibody, which is linked to a VL region of a second antibody, wherein the VL region of the second antibody is then linked to the amino terminus of an IL-10 monomer or a variant thereof; and (B) A second peptide comprising an IL-10 monomer or a variant thereof, which is linked to the VH region of a second antibody, and then the VH region of the second antibody is linked to the VL region of a first antibody.

[0432] 232. A method of treating non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD) in a patient in need, the method comprising administering to the patient a NASH or NAFLD amount of a biantibody, the biantibody comprising a first peptide chain and a second peptide chain, the first peptide chain having a heavy chain variable region (VH) from the first antibody, a light chain variable region (VL) from the second antibody, and a monomeric IL-10; the second peptide chain having VH and VL from the second antibody, and a monomeric IL-10 molecule, wherein the VH region of the first antibody binds to the VL region of the first antibody, and the VH region of the second antibody binds to the VL region of the second antibody, thereby allowing the monomeric IL-10 molecule on each peptide chain to form a functional IL-10 dimer.

[0433] 233. The method according to any of the foregoing embodiments, wherein the monomeric IL-10 is a homolog of Epstein Barr virus (EBV) IL-10 of SEQ ID No.: 3.

[0434] 234. The method according to any of the foregoing embodiments, wherein the EBV IL-10 contains an amino acid substitution at position 31 of SEQ ID No.: 3.

[0435] 235. The method according to any of the foregoing embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

[0436] 236. The method according to any of the foregoing 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.

[0437] 237. The method according to any of the foregoing embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID No.: 24 or 50.

[0438] 238. The method according to any of the foregoing embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID No.: 25 or 51.

[0439] 239. The method according to any of the foregoing embodiments further includes a connector located between the VH region and the VL region.

[0440] 240. The method according to any of the foregoing embodiments, wherein VH and VL each comprise one or more amino acid substitutions that reduce antigenicity in the patient.

[0441] 241. The method according to any of the foregoing 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.

[0442] 242. A method of treating inflammation in a patient in need, the method comprising administering an anti-inflammatory amount of a biantibody to the patient, the biantibody comprising a first peptide chain and a second peptide chain, the first peptide chain having a heavy chain variable region (VH) from the first antibody, a light chain variable region (VL) from the second antibody, and a monomeric IL-10; the second peptide chain having VH and VL from the second antibody, and a monomeric IL-10 molecule, wherein the VH region of the first antibody binds to the VL region of the first antibody, and the VH region of the second antibody binds to the VL region of the second antibody, thereby allowing the monomeric IL-10 molecule on each peptide chain to form a functional IL-10 dimer.

[0443] 243. The method according to any of the foregoing embodiments, wherein the monomeric IL-10 is a homolog of Epstein Barr virus (EBV) IL-10 of SEQ ID No.: 3.

[0444] 244. The method according to any of the foregoing embodiments, wherein the EBV IL-10 contains an amino acid substitution at position 75 of SEQ ID No.: 3.

[0445] 245. The method according to any of the foregoing embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

[0446] 246. The method according to any of the foregoing 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.

[0447] 247. The method according to any of the foregoing embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID No.: 26, 41 or 48.

[0448] 248. The method according to any of the foregoing embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID No.: 27, 42, 49.

[0449] 249. The method according to any of the foregoing embodiments further includes a connector located between the VH region and the VL region.

[0450] 250. The method according to any of the foregoing embodiments, wherein VH and VL each comprise one or more amino acid substitutions that reduce antigenicity in the patient.

[0451] 251. The method according to any of the foregoing 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.

[0452] 252. A fusion protein of formula (I-VII)

[0453] 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 (Equation VII); or any combination thereof, in “IL-10” is a monomeric sequence selected from SEQ ID No: 1, 3, 14, 18, 15, 19, 16, 20, 55, 57 or 59; more preferably, “IL-10” is composed of SEQ ID No: 55, 57 or 59; L 1 "It is a connector of SEQ ID No: 31 or 54; L 2 "It is the connector of SEQ ID No: 30; “X 1 "This is from the VH region of the first antibody, which is specific for the following: 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 the VL region obtained from the same antibody as X1; “Y1” is derived from the VH region of the second antibody, which is specific for the following: 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 the VL region obtained from the same antibody as Y1; Where X and Y come from the same or different antibodies; “Z” represents cytokines selected from IL-10, IL-10 variants, 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.

[0454] 253. The fusion protein according to the foregoing embodiments, wherein formulas II and III are capable of forming a fusion protein complex, wherein the IL-10 monomers from each of formulas II and III are capable of forming a functional homodimer IL-10 or a variant thereof.

[0455] 254. The fusion protein according to any of the foregoing embodiments, wherein Formula II is SEQ ID No: 24, 26, 28, 41, 48 or 50.

[0456] 255. The fusion protein according to any of the foregoing embodiments, wherein Formula III is SEQ ID No: 25, 27, 29, 42, 49 or 51.

[0457] 256. A fusion protein according to any of the foregoing embodiments, wherein a 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.

[0458] 257. A fusion protein according to any of the foregoing embodiments, wherein formulas IV and V are capable of forming a fusion protein complex, wherein an IL-10 monomer from each of formulas IV and V is capable of forming a functional homodimer IL-10 or a variant thereof.

[0459] 258. The fusion protein according to any of the foregoing embodiments, wherein formula IV is SEQ ID No: 35, 38, 46, 48 or 50.

[0460] 259. The fusion protein according to any of the foregoing embodiments, wherein formula V is SEQ ID No: 36, 39, 47, 49 or 51.

[0461] 260. A fusion protein according to any of the foregoing embodiments, wherein a fusion protein complex is formed between SEQ ID No: 35 and 36; 38 and 39, 46 and 47; 48 and 49; or 50 and 51.

[0462] 261. A fusion protein according to any of the foregoing embodiments, wherein formulas VI and VII are capable of forming a fusion protein complex, wherein the IL-10 monomers from each of formulas VI and VII are capable of forming a functional homodimer IL-10 or a variant thereof.

[0463] 262. The fusion protein according to any of the foregoing embodiments, wherein Formula I is SEQ ID No: 33-34, 40, 43-44, 45, 52, 53, 61, 63, 65 or 67.

[0464] 263. The fusion protein according to any of the foregoing embodiments, wherein “n” ≥ 1, and Z is IL-2, IL-7, IL-12, IL-15 or any combination thereof.

[0465] 264. The fusion protein according to any of the foregoing embodiments, wherein Z is conjugated to X 1 Y 1 Or the N-terminus of both.

[0466] 265. A method of treating cancer, the method comprising administering to a patient in need a composition comprising a fusion protein according to any of the foregoing embodiments.

[0467] 266. The method according to any of the foregoing embodiments, wherein the fusion protein is SEQ ID No: 28-29, 35-36, 38-39, 46-47, 52, 53, 61, 63, 65 or 67.

[0468] 267. The method according to any of the foregoing embodiments, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID No: 28 and 29; 35 and 36; 38 and 39; or 46 and 47.

[0469] 268. The method according to any of the foregoing embodiments, wherein the composition comprises a fusion protein of SEQ ID No: 33, 34, 44, 52 or 53, 61, 63, 65 or 67.

[0470] 269. The method according to any of the foregoing embodiments, wherein the fusion protein comprises IL-10 consisting of DV07 of SEQ ID No. 59.

[0471] 270. A method of treating an inflammatory disease, the method comprising administering to a patient in need a composition comprising a fusion protein according to any of the foregoing embodiments.

[0472] 271. The method according to any of the foregoing embodiments, wherein the fusion protein is SEQ ID No: 26-27, 41-42, 48 or 49.

[0473] 272. The method according to any of the foregoing embodiments, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID No: 26 and 27; 41 and 42; 48 and 49.

[0474] 273. The method according to any of the foregoing embodiments, wherein the composition comprises a fusion protein of SEQ ID No: 37, 40 or 43.

[0475] 274. The method of claim 18, wherein the fusion protein comprises IL-10 consisting of DV06 of SEQ ID No. 57.

[0476] 275. A method for treating lipid-based diseases, the method comprising administering to a patient in need a composition comprising a fusion protein according to any of the foregoing embodiments.

[0477] 276. The method according to any of the foregoing embodiments, wherein the fusion protein is SEQ ID No: 24-25, 50 or 51.

[0478] 277. The method according to any of the foregoing embodiments, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID No: 24 and 25; and 50 and 51.

[0479] 278. The method according to any of the foregoing embodiments, wherein the composition comprises the fusion protein of SEQ ID No:45.

[0480] References

[0481] Asadullah, K.(1999). Interleukin 10 treatment of psoriasis: clinicalresults of a phase 2 trial. Archives Dermantology, 187-192.

[0482] Berman, RM(1996). Systemic Administration of Cellular IL-10Induces an Effective Specific and Long-lived Immune Response AgainstEstablished Tumors in Mice. Journal of Immunology, 231-238.

[0483] Blum, AM(2003). CD4+ T cells from IL-10-deficient mice transfersusceptibility to NSAID-induced Rag colitis. American Journal of Physiology,G320 - G325.

[0484] Chan, I. H.(2015). The Potentiation of IFNg and Induction ofCytotoxic Proteins by Pegylated IL-10 in Human CD8 T cells. Journal ofInterferon and Cytokine Research.

[0485] Chan, I. H.(2016). PEG-rIL-10 treatement decreases FoxP3+ T regsdespite upregulation of intratumoral IDO. OncoImmunology.

[0486] Chan, I. H.(2016). PEGylated IL-10 Activates Kupffer Cells to ControlHypercholesterolemia. PLOS One.

[0487] Chernoff, A. E.(1995). A randomized, controlled trial of IL-10 inhumans. Inhibition of inflammatory cytokine production and immune responses.Journal of Immunology, 5492-5499.

[0488] Conaway, E. A.(2017). Inhibition of Inflammatory Gene Transcriptionby IL-10 is Associated with Rapid Suppression of Lipopolysaccharide InducedEnhancer Activation. Journal of Immunology.

[0489] Correa, I.(2009). Defective IL-10 production in severe phenotypes ofCrohn's disease. Journal of Leukocyte Biology, 896-903.

[0490] Correa, I.(2009). Defective IL-10 production in severe phenotypes ofCrohn's disease. Journal of Leukocyte Biology.

[0491] Emmerich, J.(2012). IL-10 directly activates and expands tumorresident CD8 + T cells without de novo infiltration from secondary lymphoidorgans. Cancer Research.

[0492] Fedorak, R. N.(2000). Recombinant Human Interleukin 10 in theTreatment of Patients With Mild to Moderately Active Crohn's DIsease.Gastroenterology, 1473-1482.

[0493] Friedrich, M.(2002). Immunomodulation by Interleukin-10 TherapyDecreases the Incidence of Relapse and Prolongs the Relapse-free Interval inPsoriasis. Journal of Investigative Dermatology, 672-677.

[0494] Fujii, S.-i.(2001). Interleukin 10 promotes the maintenance ofantitumor CD8 + T cell effector function in situ. Blood, 2143-2151.

[0495] Ghosh, S.(2006). Interferring with interferons in inflammatory boweldiseasse. Gut.

[0496] Hsu, D.-H.(1990). Expression of Interleukin-10 Activity by Epstein-Barr Virus Protein BCRF1. Science, 830-832.

[0497] Jones, B. C.(2002). Crystal structure of human cytomegalovirus IL-10bound to soluble human IL-10R1. PNAS, 9404-9409.

[0498] Kennedy Norton(2008). IL-10 Suppresses Mast Cell IgE ReceptorExpression and Signaling In Vitro and In Vivo. Journal of Immunology, 2848-2854.

[0499] Kimple et al(2013). Overview of Affinity Tags for ProteinPurification. Curr. Protoc. Protein Sci., 73:Unit 9.9

[0500] Kuhn, R.(1993). Interleukin-10 Deficient Mice Develop ChronicEnterocolitis. Cell, 263-274.

[0501] Kundu, N.(1997). Interleukin 10 Inhibits Tumor MetastasisDownregulates MHC Class I and Enhances NK Lysis. Cellualar Immunology, 55-61.

[0502] Lauw, F. N.(2000). Proinflammatory Effects of IL-10 During HumanEndotoxemia. Journal of Immunology, 2783-2789.

[0503] Leach, M. W.(1999). The Role of IL-10 in Inflammatory Bowel DIsease:"Of Mice and Men". Toxicilogic Pathology, 123-133.

[0504] Liu, Y.(1997). The EBV IL-10 homologue is a selective agonist withimpaired binding to the IL-10 receptor. Journal of Immunology, 604-613.

[0505] Maini, R. N.(1997). rHuIL-10 in subjects with active rheumatoidarthritis(RA): A phase I and cytokine response study. Arthritis Rheumatology,40.

[0506] Malefyt, R. d.(1991). Interleukin 10 Inhibits Cytokine Synthesis byHuman Monocytes An Autoregulatory Role of IL-10 Produced by Monocytes. J.Exp. Med., 1209-1220.

[0507] Malefyt, R. d.(1993). Direct effects of IL-10 on subsets of human CD4T cell clones and resting T cells - Specific inhibition of IL-2 productionand proliferation. Journal of Immunology, 4754-4765.

[0508] Minter, R. M.(2001). Adenoviral Delivery of Human and Viral IL-10 inMurine Sepsis. Journal of Immunology, 1053-1-59.

[0509] Mumm, J. B.(2011). IL-10 Elicits IFNg Dependent Tumor ImmuneSurveillance. Cancer Cell, 781-796.

[0510] Naing, A.(2016). Safety, Antitumor Activity, and Immune Activation ofPegylated Recombinant Human Interleukin-10(AM0010) in Patients With AdvancedSolid Tumors. Journal of Clinical Oncology.

[0511] Naing, A.(2018). PEGylated IL-10(Pegilodecakin) Induces SystemicImmune Activation, CD8 + T Cell Invigoration and Polyclonal T Cell Expansionin Cancer Patients. Cancer Cell.

[0512] Ouyang, P.(2014). IL-10 encoded by viruses: a remarkable example ofindepenent acquisition of a cellular gene by viruses and it's subsequentevolution in the viral genome. Journal of General Virology, 245-262.

[0513] Salek-Ardakani, S.(2002). Epstein-Barr Virus Encoded Interleukin-10Inhibits HLA-Class I, ICAM-1, and B7 Expression on Human Monocytes:Implications for Immune Evasion by EBV. Virology, 342-351.

[0514] Sasaki, T.(1992). The role of interferon g in the pathogenesis ofCrohn's disease. Gastroenterologia Japanica.

[0515] Schreiber, S.(2000). Safety and Efficacy of Recombinant HumanInterleukin 10 in Chronic Active Crohn's Disease. Gastroenterology, 1461-1472.

[0516] Slobedman, B.(2009). Virus-Encoded Homologs of Cellular Interleukin-10 and Their Control of Host Immune Function. Journal of Virology, 9618-9629.

[0517] Strober, W.(2011). Pro-Inflammatory Cytokines in the Pathogenesis ofIBD. Gastroenterology, 1756-1767.

[0518] Thompson-Snipes, L.(1991). Interleukin 10: A Novel Stimulatory Factorfor Mast Cells and Their Progenitors. Journal of Experimental Medicine, 507-510.

[0519] Tilg, H.(2002). Treatment of Crohn's disease with recombinant humaninterleukin 10 induces the proinflammatory cytokine interferon gamma. Gut,191-195.

[0520] Vieira, P.(1991). Isolation and expression of human cytokinesynthesis inhibitory factor cDNA clones Homology to Epstein Barr virus openreading frame BCRF1. PNAS, 1172-1176.

[0521] Yoon, S. I.(2005). Same Structure Different Function CrystalStructure of the Epstein Barr Virus IL-10 Bound to the Soluble IL-10R1 Chain.Structure, 551-564.

[0522] Yoon, S. I.(2012). Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties. Journal of BiologicalChemistry.

[0523] Zheng, L.(1996). Interleukin 10 Inhibits Tumor Metastasis Through anNK Cell-dependent Mechanism. Journal of Experimental Medicine, 579-584.

[0524] Zhu, L.(2017). IL-10 and IL-10 Receptor Mutations in Very Early OnsetInflammatory Bowel Disease. Gastroenterology Research, 65-69.

[0525] Zigmond, E.(2014). Macrophage-Restricted Interkeukin-10 ReceptorDeficiency, but Not IL-10 Deficiency, Causes Severe Spontaneous Colitis.Cell.

Claims

1. A fusion protein of formula (I) IL10-L 1 -X 1 -L 1 -X 2 -L 1 -IL10 (type I); in "IL-10" is a monomeric sequence selected from SEQ ID No: 1, 3, 14, 15, 16, 18, 19, 55, 57 or 59; L 1 "It is a connector of SEQ ID No: 31 or 54; L 2 "It is the connector of SEQ ID No: 30; X 1 "This is from the VH region of the first antibody, which is specific for the following: 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 "The VL region was obtained from the same antibody as X1; Y 1 "This is from the VH region of the second antibody, which is specific for the following: 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 "The VL region was obtained from the same antibody as Y1; Where X and Y come from the same or different antibodies; "Z" stands for cytokine, which is 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.

2. The fusion protein according to claim 1, wherein formula I is SEQ ID No: 33-34, 40, 43-44, 45, 52 or 53.

3. Use of the composition of claim 1 in the preparation of a medicament for treating cancer, comprising administering the composition comprising the fusion protein of claim 1 to a patient in need.

4. The use according to claim 3, wherein the fusion protein is SEQ ID No: 33-34, 40, 43-44, 45, 52 or 53.

5. The use according to claim 3, wherein the fusion protein comprises IL-10 consisting of DV07 of SEQ ID No.

59.

6. Use of the composition of claim 1 in the preparation of a medicament for treating inflammatory diseases, said use comprising administering the composition comprising the fusion protein of claim 1 to a patient in need.

7. The use according to claim 6, wherein the composition comprises a fusion protein of SEQ ID No: 37, 40 or 43.

8. The use according to claim 6, wherein the fusion protein comprises IL-10 consisting of DV06 of SEQ ID No.

57.

9. Use of the composition of claim 1 in the preparation of a medicament for treating lipid-based diseases, said use comprising administering the composition comprising the fusion protein of claim 1 to a patient in need.

10. The use according to claim 9, wherein the composition comprises the fusion protein of SEQ ID No:45.

Images