Immunomodulatory fusion protein

Abstract

To provide immunomodulatory fusion proteins.SOLUTION: An isolated fusion protein comprises: (i) a portion of an IL-10 receptor; (ii) an amino acid linker; and at least one of (iii) a portion of an extracellular domain, transmembrane domain or intracellular domain of a cytokine, cytokine receptor or immunomodulatory protein, (iv) an immunoglobulin (Ig) hinge region, and (v) an immunoglobulin (Ig) Fc domain.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 564,145, filed on September 27, 2017, which is incorporated herein by reference in its entirety.

[0002] Field of the Invention The field of the present invention is molecular biology, specifically immunology and fusion proteins, such as cytokine receptor fusion proteins.

Background Art

[0003] Background Cytokines are small, secreted cell signaling proteins that have a wide range of activities including the control of cell growth and differentiation and the regulation of immune function. Cytokines, cytokine receptors, and certain other immunomodulatory proteins are used as therapeutic agents for treating various medical conditions. However, administration of such proteins, for example, by subcutaneous or vascular routes, can result in inappropriate cellular and extracellular localization, thereby limiting therapeutic activity and / or increasing the risk of toxicity.

[0004] IL-10 is a homodimeric cytokine with immunoregulatory properties produced by cells including activated Th2 cells, B cells, keratinocytes, monocytes, and macrophages (Non-Patent Document 1). IL-10 inhibits the activation and effector functions of various cells including T cells, monocytes, and macrophages. In particular, IL-10 inhibits cytokine synthesis, including the synthesis of IL-1, IFN-γ, and TNF by cells such as Th1 cells, natural killer cells, monocytes, and macrophages (Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7). A plurality of pathogens, including intracellular pathogens, induce IL-10 production to delay or avoid (stall) the effective elimination of the pathogen by the immune system (Moore et al. (1993) supra).

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

[0006] Despite the progress made to date in the treatment of IL-10-mediated disorders, there is a need for improved therapies for treating such disorders. MEANS FOR SOLVING THE PROBLEM

[0007] SUMMARY OF THE INVENTION The present invention is based, in part, on the discovery of linker sequences that improve the function of fusion proteins, such as cytokine receptor fusion proteins, such as IL-10 receptor (IL-10R) fusion proteins, such as IL-10 receptor subunit α (IL-10RA) fusion proteins, such as IL-10 traps. The linker sequence can optimally bind the ligand-binding portion of the fusion protein (e.g., cytokine receptor) to the ligand (e.g., cytokine), provide temporal and spatial co-localization of two or more components of the fusion protein (e.g., the two subunits of a dimeric cytokine), optimize expression from an expression vector (e.g., viral vector), reduce immunogenicity, or provide a cleavage site that allows release of the components of the fusion protein. For example, the linker sequence can provide sufficient flexibility for the ligand-binding domain of the cytokine receptor to adopt its native conformation in the context of the fusion protein and can minimize the potential immunogenicity of the fusion protein for use as a therapeutic agent.

[0008] In one aspect, the present invention provides an isolated fusion protein comprising, for example, in the N-terminal to C-terminal direction: a first portion of an extracellular domain, transmembrane domain or intracellular domain of a cytokine, cytokine receptor or immunomodulatory protein; an amino acid linker; and a second portion of an extracellular domain, transmembrane domain or intracellular domain of a cytokine, cytokine receptor or immunomodulatory protein; an immunoglobulin (Ig) hinge region; and at least one of an immunoglobulin (Ig) Fc domain. In certain embodiments, the linker comprises from about 5 to about 40 amino acid residues. In certain embodiments, the fusion protein comprises a portion of an IL-10 receptor, such as the human IL-10 receptor, such as IL-10RA.

[0009] In another aspect, the present invention provides an isolated fusion protein comprising, in the N-terminal to C-terminal direction: a soluble portion of an extracellular domain of a cytokine receptor; an amino acid linker; an immunoglobulin (Ig) hinge region; and an immunoglobulin (Ig) Fc domain, wherein the linker comprises from about 5 to about 40 amino acid residues. In certain embodiments, the cytokine receptor is an IL-10 receptor, such as the human IL-10 receptor, such as IL-10RA.

[0010] In certain embodiments of any of the foregoing fusion proteins, the amino acid linker can comprise, for example, from about 5 to about 15, from about 5 to about 20, from about 5 to about 30, from about 10 to about 15, from about 10 to about 20, from about 10 to about 30, from about 10 to about 40, from about 15 to about 20, from about 15 to about 30 or from about 15 to about 40 amino acid residues.

[0011] In certain embodiments of any of the foregoing fusion proteins, the amino acid linker sequence is derived from an endogenous human protein, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM, albumin or casein. In certain embodiments, the amino acid linker comprises the C-terminal portion of an immunoglobulin (Ig) CH1 domain, such as the CH1 domain of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM. In certain embodiments, the amino acid linker comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:57. In certain embodiments, the amino acid linker comprises the C-terminal portion of the IgG1 CH1 domain, such as the amino acid linker comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:57, such as the amino acid sequence of SEQ ID NO:57.

[0012] In certain embodiments of any of the foregoing fusion proteins, the amino acid linker comprises a sequence derived from a cytokine, a signaling molecule, an immunomodulatory protein or peptide or a biologically active peptide.

[0013] In certain embodiments of any of the foregoing fusion proteins, the amino acid linker comprises a cleavage site, such as a proteolytic cleavage site, such as a proteolytic cleavage site cleaved by a protease present in the endoplasmic reticulum or Golgi of eukaryotic cells. In certain embodiments, the proteolytic cleavage site is a furin cleavage site, such as a furin cleavage site comprising the sequence RX1X2R (SEQ ID NO:50) (wherein X1 is any amino acid and X2 is Lys or Arg), such as a furin cleavage site comprising the sequence RAKR (SEQ ID NO:51). In certain embodiments of any of the foregoing fusion proteins, the amino acid linker is proteolytically stable in mammals or plants.

[0014] In certain embodiments of any of the foregoing fusion proteins, the soluble portion of the extracellular domain of the cytokine receptor is the soluble portion of the extracellular domain of a human IL-10R, such as the extracellular domain of IL-10RA. For example, in certain embodiments, the soluble portion of the extracellular domain of the cytokine receptor comprises the amino acid sequence of SEQ ID NO: 12 or amino acid residues 22 to 229 of SEQ ID NO: 12.

[0015] In certain embodiments of any of the foregoing fusion proteins, the fusion protein comprises one or more of IL-10, TGF-β, TGFβ receptor, such as TGFβ type II receptor (TβRII), CD80, CD19, CD20, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12B / p40, IL-23A / p19, IL27A / p28, IL-27B / EBI3, IL-15, CD154, CD70, TNF-α, CD86, CD137, CD137L, BORIS / CTCFL, FGF, ICAM, IL-24, GM-CSF, MAGE, NY-ESO-1, angiostatin, endostatin, acetylcholine, interferon-γ, DKK1 / Wnt, p53, Ox40L, GM-CSF, IL-15 receptor fusion protein, GITRL, CD40L, CD70, secreted flagellin, IL-12, thymidine kinase, anti-PD-1 antibody heavy or light chain, anti-PD-L1 antibody heavy or light chain and anti-CTLA-4 antibody heavy or light chain or functional fragments thereof.

[0016] In certain embodiments of any of the foregoing fusion proteins, the Ig hinge region is selected from the hinge regions of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM, and the Ig Fc domain is selected from the Fc domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM. In certain embodiments, the Ig hinge region and the Fc domain together comprise an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21. In certain embodiments, the Ig Fc, Ig hinge region and Ig CH1 domain are derived from a single immunoglobulin.

[0017] In certain embodiments of any of the foregoing fusion proteins, the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 58. In certain embodiments, the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 58. In certain embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 58.

[0018] In certain embodiments of any of the foregoing fusion proteins, the fusion protein comprises an amino acid sequence having a sequence identity higher than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to a sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 58.

[0019] In another aspect, the present invention provides a dimeric cytokine binding protein comprising any two of the aforementioned fusion proteins that covalently bind together, wherein each fusion protein comprises an extracellular domain of a cytokine receptor, and the two extracellular domains together define a binding site for a cytokine.

[0020] In another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding any of the aforementioned fusion proteins.

[0021] In another aspect, the present invention provides an expression vector comprising any of the aforementioned nucleic acids.

[0022] In another aspect, the present invention provides a host cell comprising any of the aforementioned expression vectors. In another aspect, the present invention provides a method for producing a fusion protein, comprising the steps of growing a host cell under conditions that express the fusion protein and purifying the fusion protein. In another aspect, the present invention provides a method for expressing a fusion protein in a target cell, comprising the step of exposing the target cell to an effective amount of any of the aforementioned expression vectors. In one embodiment, the fusion protein is cleaved into two polypeptide chains after translation.

[0023] In another aspect, any of the aforementioned fusion proteins or expression vectors can be used, for example, to reduce cytokine activity in a subject, thereby treating various medical symptoms mediated by cytokines, such as IL-10. In another aspect, any of the aforementioned fusion proteins or expression vectors can be used to inhibit the growth of tumor cells in vitro and / or in vivo, to inhibit tumor growth in a subject in need of such inhibition, or to treat cancer in a subject in need of cancer treatment. The subject can be, for example, an animal, such as a mammal, such as a human, such as a pediatric human. For example, when administered to a human subject having cancer, the fusion protein or expression vector inhibits or reduces tumor growth or reduces tumor burden in the subject.

[0024] In certain embodiments, the cancer can be selected from melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, head and neck cancer, breast cancer, anal cancer, cervical cancer, non-small cell lung cancer, mesothelioma, small cell lung cancer, renal cell carcinoma, prostate cancer, gastroesophageal cancer, colorectal cancer, testicular cancer, bladder cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, cancers of the brain and central nervous system, thyroid cancer, parathyroid cancer (e.g., parathyroid carcinoma), endometrial cancer, neuroendocrine cancer, lymphoma (e.g., Hodgkin and non-Hodgkin), leukemia, Merkel cell carcinoma, gastrointestinal stromal tumor, multiple myeloma, uterine cancer, sarcoma, kidney cancer, eye cancer, pancreatic cancer, and germ cell cancer (e.g., ovarian germ cell cancer). In certain embodiments, the cancer can be selected from leukemia, breast cancer, lung cancer, pancreatic cancer, endometrial cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, colorectal cancer, gastric cancer, head and neck cancer, and leukemia.

[0025] In certain embodiments, the fusion protein or expression vector is administered in combination with one or more treatments selected from surgery, radiation, chemotherapy, immunotherapy, hormone therapy, and viral therapy. In certain embodiments, the fusion protein or expression vector is administered in combination with lymphocytes, such as T cells, such as CAR T cells.

[0026] Any of the foregoing fusion proteins or expression vectors can also be used to treat an inflammatory condition or infection in a subject in need thereof.

[0027] These and other aspects and advantages of the invention are set forth in the following drawings, detailed description, and claims.

[0028] That is, the gist of the present invention relates to the following. Item 1 (i) a portion of the IL-10 receptor; and (ii) an amino acid linker; and (iii) a portion of the extracellular, transmembrane, or intracellular domain of a cytokine, cytokine receptor, or immunomodulatory protein; (iv) Immunoglobulin (Ig) hinge region; or (v) Immunoglobulin (Ig) Fc domain At least one of an isolated fusion protein. Item 2 The isolated fusion protein according to item 1, wherein the linker contains about 5 to about 40 amino acid residues. Item 3 In the N-terminal to C-terminal direction, (i) Soluble portion of the extracellular domain of the IL-10 receptor; (ii) Amino acid linker; (iii) Immunoglobulin (Ig) hinge region; and (iv) Immunoglobulin (Ig) Fc domain An isolated fusion protein containing, wherein the linker contains about 5 to about 40 amino acid residues. Item 4 The isolated fusion protein according to any one of items 1 to 3, wherein the linker contains about 5 to about 30 amino acid residues. Item 5 The isolated fusion protein according to any one of items 1 to 3, wherein the linker contains about 5 to about 20 amino acid residues. Item 6 The isolated fusion protein according to any one of items 1 to 3, wherein the linker contains about 5 to about 15 amino acid residues. Item 7 The isolated fusion protein according to any one of items 1 to 3, wherein the linker contains about 10 to about 40 amino acid residues. Item 8 The isolated fusion protein according to any one of items 1 to 3, wherein the linker contains about 10 to about 30 amino acid residues. Item 9 The isolated fusion protein according to any one of items 1 to 3, wherein the linker contains about 10 to about 20 amino acid residues. Item 10 The isolated fusion protein according to any one of items 1 to 3, wherein the linker contains about 10 to about 15 amino acid residues. Item 11 The isolated fusion protein according to any one of claims 1 to 10, wherein the linker comprises a sequence derived from an endogenous human protein. Claim 12 The isolated fusion protein according to any one of claims 1 to 11, wherein the linker comprises the C-terminal portion of the immunoglobulin (Ig) CH1 domain. Claim 13 The isolated fusion protein according to claim 12, wherein the Ig CH1 domain is selected from the CH1 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM. Claim 14 The isolated fusion protein according to claim 13, wherein the linker comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:57. Claim 15 The isolated fusion protein according to claim 14, wherein the Ig CH1 domain is the IgG1 CH1 domain. Claim 16 The isolated fusion protein according to claim 15, wherein the linker comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:57. Claim 17 The isolated fusion protein according to claim 16, wherein the linker comprises the amino acid sequence of SEQ ID NO:57. Claim 18 The isolated fusion protein according to any one of claims 1 to 11, wherein the linker comprises a sequence derived from a human protein selected from albumin and casein. Claim 19 The isolated fusion protein according to claim 18, wherein the linker comprises an amino acid sequence selected from SEQ ID NO:10 and SEQ ID NO:11. Claim 20 The isolated fusion protein according to any one of claims 1 to 11, wherein the linker comprises a sequence derived from a cytokine, a signal transduction molecule, an immunomodulatory protein or an immunomodulatory peptide. Claim 21 An isolated fusion protein according to any one of claims 1 to 20, wherein the linker contains a cleavage site. Claim 22 The isolated fusion protein according to claim 21, wherein the cleavage site is a proteolytic cleavage site. Claim 23 The isolated fusion protein according to claim 22, wherein the proteolytic cleavage site is cleaved by a protease present in the endoplasmic reticulum or Golgi apparatus of eukaryotic cells. Claim 24 The isolated fusion protein according to claim 22 or 23, wherein the proteolytic cleavage site is a furin cleavage site. Claim 25 The isolated fusion protein according to claim 24, wherein the furin cleavage site contains RX1X2R (SEQ ID NO: 50) (wherein X1 is any amino acid and X2 is Lys or Arg). Claim 26 The isolated fusion protein according to claim 22, wherein the furin cleavage site contains RAKR (SEQ ID NO: 51). Claim 27 An isolated fusion protein according to any one of claims 1 to 20, wherein the linker contains an amino acid sequence that is stable to proteolysis in mammals or plants. Claim 28 The isolated fusion protein according to any one of claims 1 to 27, wherein the IL-10 receptor is a human IL-10 receptor. Claim 29 The isolated fusion protein according to claim 28, wherein the soluble portion of the extracellular domain of the IL-10 receptor contains the amino acid sequence of SEQ ID NO: 12. Claim 30 The isolated fusion protein according to claim 28, wherein the soluble portion of the extracellular domain of the IL-10 receptor contains amino acid residues 22 to 229 of SEQ ID NO: 12. Claim 31 The isolated fusion protein according to any one of claims 1 to 30, wherein the Ig Fc domain and the Ig hinge region are selected from the Fc domains and hinge regions of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Claim 32 The isolated fusion protein according to item 31, wherein the Ig Fc domain and the Ig hinge region comprise an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. Item 33 The isolated fusion protein according to item 32, wherein the Ig Fc domain and the Ig hinge region are a human IgG1 Fc domain and a human IgG1 hinge region. Item 34 The isolated fusion protein according to item 33, wherein the Ig Fc domain and the Ig hinge region comprise the amino acid sequence of SEQ ID NO: 13. Item 35 The isolated fusion protein according to any one of items 1 to 34, wherein the Ig Fc, the Ig hinge region, and the Ig CH1 domain are derived from a single immunoglobulin. Item 36 The isolated fusion protein according to any one of items 1 to 35, comprising an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 58. Item 37 The isolated fusion protein according to item 36, comprising an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 58. Item 38 The isolated fusion protein according to item 37, comprising the amino acid sequence of SEQ ID NO: 58. Item 39 A cytokine-binding protein comprising two fusion proteins according to any one of items 1 to 38, wherein each fusion protein comprises an extracellular domain of a cytokine receptor, the two fusion proteins are covalently linked together, and the two extracellular domains together define a binding site for binding to a cytokine. Item 40 An isolated nucleic acid comprising a nucleotide sequence encoding the fusion protein according to any one of Items 1 to 39. Item 41 An expression vector comprising the nucleic acid according to Item 40. Item 42 A host cell comprising the expression vector according to Item 41. Item 43 (a) A step of growing the host cell according to Item 42 under conditions for expressing the fusion protein; and (b) A step of purifying the fusion protein A method for producing a fusion protein, comprising: Item 44 (i) The fusion protein according to any one of Items 1 to 39 or the expression vector according to Item 41; and (ii) A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier or diluent. Item 45 A method for expressing a fusion protein in a target cell, comprising a step of exposing the target cell to an effective amount of the expression vector according to Item 41 to express the fusion protein. Item 46 The method according to Item 45, wherein the fusion protein is cleaved into two polypeptide chains after translation. Item 47 A method for inhibiting the growth of tumor cells, comprising a step of exposing the tumor cells to an effective amount of the dimeric fusion protein according to Item 39 to inhibit the growth of the tumor cells. Item 48 A method for inhibiting the growth of tumor cells, comprising a step of exposing the tumor cells to an effective amount of the fusion protein according to any one of Items 1 to 38 to inhibit the growth of the tumor cells. Item 49 A method for inhibiting tumor growth in a subject in need thereof, comprising a step of administering an effective amount of the dimeric fusion protein according to Item 39 to the subject in need of inhibiting tumor growth to inhibit the growth of the tumor. Item 50 A method for inhibiting tumor growth in a subject in need thereof, comprising a step of administering an effective amount of the fusion protein according to any one of Items 1 to 38 to the subject in need of inhibiting tumor growth to inhibit the growth of the tumor. Item 51 A method for treating cancer in a subject in need of cancer treatment, comprising the step of administering to the subject an effective amount of the dimeric fusion protein according to item 39. Item 52 A method for treating cancer in a subject in need of cancer treatment, comprising the step of administering to the subject an effective amount of the fusion protein according to any one of items 1 to 38. Item 53 A method for reducing IL-10 activity in a cell, comprising the step of exposing the cell to an effective amount of the dimeric fusion protein according to item 39 to reduce IL-10 activity. Item 54 A method for reducing IL-10 activity in a cell, comprising the step of exposing the cell to an effective amount of the fusion protein according to any one of items 1 to 38 to reduce IL-10 activity. Item 55 A method for treating an inflammatory condition in a subject in need of treatment of the inflammatory condition, comprising the step of administering to the subject an effective amount of the dimeric fusion protein according to item 39. Item 56 A method for treating an inflammatory condition in a subject in need of treatment of the inflammatory condition, comprising the step of administering to the subject an effective amount of the fusion protein according to any one of items 1 to 38. Item 57 A method for inhibiting the growth of tumor cells, comprising the step of exposing the tumor cells to an effective amount of the expression vector according to item 41 to inhibit the growth of the tumor cells. Item 58 A method for inhibiting tumor growth in a subject in need of inhibition of tumor growth, comprising the step of administering to the subject an effective amount of the expression vector according to item 41 to inhibit the growth of the tumor. Item 59 A method for treating cancer in a subject in need of cancer treatment, comprising the step of administering to the subject an effective amount of the expression vector according to item 41. Item 60 A method for reducing IL-10 activity in a cell, comprising exposing the cell to an effective amount of the expression vector according to item 41 to reduce IL-10 activity. Item 61 A method for treating an inflammatory condition in a subject in need thereof, comprising administering to the subject in need of treatment for an inflammatory condition an effective amount of the expression vector according to item 41. Item 62 A method for treating an infection in a subject in need thereof, comprising administering to the subject in need of treatment for an infection an effective amount of the expression vector according to item 41. Item 63 The method according to item 51, 52 or 59, wherein the cancer is selected from melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, head and neck cancer, breast cancer, anal cancer, cervical cancer, non-small cell lung cancer, mesothelioma, small cell lung cancer, renal cell carcinoma, prostate cancer, gastroesophageal cancer, colorectal cancer, testicular cancer, bladder cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, cancer of the brain and central nervous system, thyroid cancer, parathyroid cancer (e.g., parathyroid carcinoma), endometrial cancer, neuroendocrine cancer, lymphoma (e.g., Hodgkin and non-Hodgkin), leukemia, Merkel cell carcinoma, gastrointestinal stromal tumor, multiple myeloma, uterine cancer, sarcoma, kidney cancer, eye cancer, pancreatic cancer and germ cell cancer (e.g., ovarian germ cell cancer). Item 64 The method according to item 51, 52 or 59, wherein the cancer is selected from leukemia, breast cancer, lung cancer, pancreatic cancer, endometrial cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, colorectal cancer, gastric cancer, head and neck cancer and leukemia. Item 65 The method according to item 64, wherein the cancer is selected from skin cancer, head and neck cancer and lung cancer. Item 66 The method according to any one of items 49 to 52, 55, 56, 58, 59 or 61 to 65, wherein the fusion protein or expression vector is administered to the subject in combination with one or more treatments selected from surgery, radiation, chemotherapy, immunotherapy, hormone therapy and viral therapy. Item 67 The method according to any one of items 49 to 52, 55, 56, 58, 59 or 61 to 65, wherein the fusion protein or expression vector is administered to the subject in combination with lymphocytes. Item 68 The method according to item 67, wherein the lymphocyte is a T cell. Item 69 The method according to item 68, wherein the T cell is a CAR T cell. Item 70 The method according to any one of items 49 to 52, 55, 56, 58, 59 or 61 to 69, wherein the subject is a human or an animal. Item 71 The method according to item 70, wherein the subject is a pediatric human.

Advantages of the Invention

[0029] According to the present invention, an immunomodulatory fusion protein can be provided.

Brief Description of the Drawings

[0030] Description of the Drawings The present invention can be more fully understood with reference to the following drawings.

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0031] Detailed Description The present invention provides recombinant fusion proteins for use in the treatment of various medical conditions in a subject, such as inhibition of tumor cell proliferation, inhibition of tumor growth, treatment of cancer, treatment of inflammatory conditions or treatment of infections. Exemplary fusion proteins include: a first portion of an extracellular domain, transmembrane domain or intracellular domain of a cytokine, cytokine receptor or immunomodulatory protein; an amino acid linker; and a second portion of an extracellular domain, transmembrane domain or intracellular domain of a cytokine, cytokine receptor or immunomodulatory protein; an immunoglobulin (Ig) hinge region; or at least one of an immunoglobulin (Ig) Fc domain. The first and second portions can be portions of the same protein or portions of different proteins, and even if they are the same protein, it is contemplated that the first and second portions can be different portions of the same protein. In one embodiment, the linker comprises from about 5 to about 40 amino acid residues. An exemplary fusion protein of the present invention is cytokine trap.

[0032] A cytokine trap, such as an IL-10 trap, is a molecule comprising a soluble portion of an extracellular domain of a cytokine receptor, such as an IL-10 receptor (IL-10R), such as the α subunit of the IL-10 receptor (IL-10RA), designed to bind to or otherwise sequester a target cytokine. In a cytokine trap, the extracellular domain of the cytokine receptor can be fused to an immunoglobulin (Ig) hinge region and an immunoglobulin (Ig) Fc domain, which can for example enable increased stability, Fc effector function and / or multimerization, such as dimerization. Dimerization resulting from fusion to the Ig hinge region and Ig Fc domain is particularly advantageous for cytokine receptors that exist as dimeric receptor complexes on the cell surface, such as TβRII.

[0033] Conventional cytokine traps, such as the IL-10 trap, comprise two polypeptide chains, each polypeptide chain comprising a soluble portion of the extracellular domain of a cytokine receptor fused to an Ig hinge region and an Ig Fc domain. The soluble portion of the extracellular domain of the cytokine receptor is typically fused directly to the Ig hinge region without an intervening sequence. The two polypeptide chains are covalently linked by a disulfide bond between cysteine residues of their respective Ig hinge regions. Each polypeptide chain provides a soluble portion of the extracellular domain of a cytokine receptor, such as the IL-10R, such as the IL-10RA, and the two soluble portions of the extracellular domain of the cytokine receptor together define a binding site for the cytokine. A schematic representation of a dimeric protein comprising a dimeric cytokine receptor, an immunoglobulin (antibody) molecule, and two covalently linked fusion proteins each comprising a soluble portion of the extracellular domain of a cytokine receptor fused to an Ig hinge region and an Ig Fc domain is shown in FIG. 1A.

[0034] The present invention is based, in part, on the discovery that conventional cytokine traps, such as the IL-10 trap, comprising fusion proteins of soluble portions of the extracellular domains of cytokine receptors with Ig hinge regions and Ig Fc domains, do not optimally bind their target cytokines. For example, conventional IL-10 traps do not provide sufficient flexibility between the two IL-10 ligand-binding domains to allow the two IL-10 ligand-binding domains to assume an optimal conformation to define an IL-10 binding site.

[0035] Accordingly, in one aspect, the present invention provides an isolated fusion protein comprising, in the N-terminal to C-terminal direction: a soluble portion of the extracellular domain of a cytokine receptor; an amino acid linker; an immunoglobulin (Ig) hinge region; and an immunoglobulin (Ig) Fc domain, wherein the linker comprises from about 5 to about 40 amino acid residues. The linker sequence enables, for example, the binding domain in the extracellular domain of the cytokine receptor to optimally bind its target cytokine. This is particularly important when the fusion protein is a dimer comprising two of the aforementioned fusion proteins that together define a binding site for the cytokine binding protein to bind to its target cytokine. Without a linker, the two binding domains may be sterically hindered from forming an optimal binding site (Figure 1B). Various features and aspects of the present invention are discussed in more detail below.

[0036] I. Fusion Protein Exemplary fusion proteins can comprise a first portion of an extracellular domain, transmembrane domain or intracellular domain of a cytokine, cytokine receptor or immunomodulatory protein; an amino acid linker; and at least one of a second portion of an extracellular domain, transmembrane domain or intracellular domain of a cytokine, cytokine receptor or immunomodulatory protein; an immunoglobulin (Ig) hinge region; and an immunoglobulin (Ig) Fc domain. For example, the disclosed fusion proteins can comprise, in the N-terminal to C-terminal direction: a soluble portion of the extracellular domain of a cytokine receptor; an amino acid linker; an immunoglobulin (Ig) hinge region; and an immunoglobulin (Ig) Fc domain, wherein the linker comprises from about 5 to about 40 amino acid residues.

[0037] Exemplary cytokines include IL-1α, IL-1β, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-α, IFN-β, IFN-γ, CD154, LT-β, TNF-α, TNF-β, 4-1BBL APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β2, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF and MSP.

[0038] As used herein, an "immunomodulatory" protein refers to a protein that modulates the function of the immune system of a subject. An immunomodulatory protein can, for example, modulate the function of B cells, T cells and / or antibody production. Exemplary immunomodulatory proteins include checkpoint inhibitors. Exemplary immunomodulatory proteins can include, for example, CTLA-4, CD70, IL-2, CD40L, OX40L, IL-12, IL-7, PD-1 or PD-L1, or any protein that modulates their activity. Further exemplary immunomodulatory proteins can include anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0039] As used herein, the "soluble portion of the extracellular domain of a cytokine receptor" refers to any extracellular domain of a cytokine receptor or a fragment of an extracellular domain of a cytokine receptor that can bind to a target cytokine. It is understood that the soluble portion of the extracellular domain of a cytokine receptor also contemplates a portion of the extracellular domain that contains a binding domain that can bind to a target cytokine either alone or in combination with a second binding domain (e.g., in the case of a dimeric fusion protein).

[0040] Exemplary cytokine receptors include type I cytokine receptors (e.g., GM-CSF receptor, G-CSF receptor, type I IL receptor, Epo receptor, LIF receptor, CNTF receptor or TPO receptor), type II cytokine receptors (e.g., IL-10 receptor, IFN-α receptor (e.g., IFNAR1 or IFNAR2), IFN-β receptor, IFN-γ receptor (e.g., IFNGR1 or IFNGR2), chemokine receptors (e.g., CC chemokine receptor, CXC chemokine receptor, CX3C chemokine receptor or XC chemokine receptor), tumor necrosis factor superfamily receptors (TNFR; e.g., TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSF1A / TNFR1 / CD120a or TNFRSF1B / TNFR2 / CD120b), TGFβ superfamily receptors (e.g., TGFβ type I receptor or TGFβ type II receptor), or immunoglobulin (Ig) superfamily receptors (e.g., interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA or PDGFRB) or SCFR). Preferred cytokine receptors include dimeric cytokine receptors, such as TGFβ superfamily receptors, such as the human TGFβ type II receptor (TβRII). In certain embodiments, the soluble portion of the extracellular domain of the cytokine receptor comprises, for example, the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12, and / or fragments thereof that include a binding domain that binds to IL-10, such as the soluble portion of the extracellular domain of human IL-10R, such as human IL-10RA.

[0041] The soluble portion of the extracellular domain of the cytokine receptor retains the ability to bind its natural ligand. In certain embodiments, the soluble portion of the extracellular domain retains at least 50%, 60%, 70%, 80%, 90% or 95% of the binding activity to its natural ligand when compared to the full-length cytokine receptor.

[0042] In certain embodiments, the fusion protein can comprise one or more of, for example, TβRII, TGF-β, CD80, CD19, CD20, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12B / p40, IL-23A / p19, IL-27A / p28, IL-27B / EBI3, IL-15, CD154, CD70, TNF-α, CD86, CD137, CD137L, BORIS / CTCFL, FGF, ICAM, IL-24, GM-CSF, MAGE, NY-ESO-1, angiostatin, endostatin, acetylcholine, interferon-γ, DKK1 / Wnt, p53, Ox40L, GM-CSF, IL-15 receptor fusion protein, GITRL, CD40L, CD70, secreted flagellin, IL-12, thymidine kinase, an anti-PD-1 antibody heavy or light chain, an anti-PD-L1 antibody heavy or light chain, and an anti-CTLA-4 antibody heavy or light chain, or functional fragments thereof.

[0043] As used herein, the term “immunoglobulin (Ig) hinge region” typically refers to the amino acid sequence that links the CH1 domain and the CH2 domain of the immunoglobulin heavy chain constant region. The Ig hinge region can contain one or more cysteine residues that can form disulfide bonds with cysteine residues in another protein chain, for example. As used herein, the term “immunoglobulin (Ig) Fc domain” refers to a fragment of the immunoglobulin heavy chain constant region that can bind to an Fc receptor. The Ig Fc domain can contain, for example, the immunoglobulin (Ig) CH2 domain and the CH3 domain. The boundaries between the Ig CH1 domain, CH2 domain, and CH3 domain are well known in the art and can be found, for example, in the PROSITE database (available on the World Wide Web at prosite.expasy.org). For clarity, an alignment of the amino acid sequences of the CH1 domain and the CH2 domain of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM is shown in FIG. 2.

[0044] In certain embodiments, the Ig hinge region is selected from the hinge regions of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM, and the Ig Fc domain is selected from the Fc domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. In certain embodiments, the Ig hinge region and the Fc domain together comprise an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. In certain embodiments, the Ig hinge region and the Fc domain together comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0045] The amino acid linker can optimally bind the ligand-binding portion of a fusion protein (such as a cytokine receptor) to a ligand (such as a cytokine), provide temporal and spatial co-localization of two or more components of a fusion protein (such as the two subunits of a dimeric cytokine), optimize expression from an expression vector (such as a viral vector), reduce immunogenicity, or provide a cleavage site that allows release of components of the fusion protein.

[0046] The amino acid linker can comprise, for example, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 5 to about 35, about 5 to about 40, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 10 to about 35, about 10 to about 40, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 15 to about 35, or about 15 to about 40 amino acid residues. The amino acids within the linker can be naturally occurring amino acids or modified amino acids.

[0047] In certain embodiments, the amino acid linker sequence is derived from an endogenous human protein, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM, albumin or casein. In certain embodiments, the amino acid linker comprises the C-terminal portion of an immunoglobulin (Ig) CH1 domain, such as the CH1 domain of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM, for example about 5 to about 40 amino acids. In certain embodiments, the amino acid linker comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64. In certain embodiments, the amino acid linker comprises a sequence having a sequence identity higher than 85%, 90%, 95%, 96%, 97%, 98% or 99% to an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64.

[0048] A protein or polypeptide "derives from" a reference protein or polypeptide if it contains an amino acid sequence that is substantially similar to all or a corresponding portion of the wild-type amino acid sequence of the reference protein or polypeptide. In certain embodiments, a protein or polypeptide that derives from a wild-type protein or polypeptide may have one or more amino acid substitutions relative to the wild-type protein or polypeptide. For example, it is contemplated that a protein or polypeptide that derives from a wild-type protein or polypeptide may have a sequence identity of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher relative to the wild-type protein or polypeptide. Further, it is contemplated that a protein or polypeptide that derives from a wild-type protein or polypeptide may contain more conservative substitutions relative to the wild-type protein or polypeptide. As used herein, the term "conservative substitution" refers to a substitution by an amino acid that is structurally similar. For example, conservative substitutions may include members of the following groups: Ser and Cys; Leu, Ile and Val; Glu and Asp; Lys and Arg; Phe, Tyr and Trp; and Gln, Asn, Glu, Asp and His. Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., the BLOSUM 62 matrix) or the PAM substitution: p matrix (e.g., the PAM 250 matrix).

[0049] In certain embodiments, the amino acid linker sequence derives from a cytokine, a signaling molecule, an immunomodulatory protein or peptide or a biologically active peptide.

[0050] Additional linker arrays contemplated include glycine and serine rich linkers such as (G4S)3 (SEQ ID NO:49). Additional exemplary linker arrays are disclosed, for example, in George et al. (2003) PROTEIN ENGINEERING 15:871-879 as well as U.S. Patent Nos. 5,482,858 and 5,525,491.

[0051] In certain embodiments, the amino acid linker may include a cleavage site, such as a proteolytic or non-proteolytic cleavage site. In certain embodiments, the proteolytic cleavage site is cleaved by a protease present in a particular tissue, organelle or intracellular compartment. In certain embodiments, the linker includes a proteolytic cleavage site and two cysteine residues that form a disulfide bond after proteolytic cleavage. In certain embodiments, the proteolytic cleavage site is cleaved by a protease selected from matrix metalloprotease (MMP), furin, PC1, PC2, PC3, cathepsin B, protease 3 and caspase 3. In certain embodiments, the cleavage site is a proteolytic cleavage site cleaved by a protease present in the endoplasmic reticulum or Golgi of eukaryotic cells. In certain embodiments, the proteolytic cleavage site is a furin cleavage site. Furin is a ubiquitously expressed protease localized to the Golgi, and furin recognizes the consensus sequence RX1X2R (SEQ ID NO:50) (wherein X1 is any amino acid and X2 is Lys or Arg) and cleaves behind the last Arg. Furin plays a biological role in the cleavage of propeptides of proteins passing through the Golgi. Thus, in certain embodiments the proteolytic cleavage site is a furin cleavage site comprising the sequence RX1X2R (SEQ ID NO:50) (wherein X1 is any amino acid and X2 is Lys or Arg), for example a furin cleavage site comprising the sequence RAKR (SEQ ID NO:51).

[0052] In certain embodiments, the Ig Fc, Ig hinge region and Ig CH1 domain are derived from a single immunoglobulin.

[0053] In certain embodiments, the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 58. In certain embodiments, the disclosed fusion protein comprises an amino acid sequence having a sequence identity higher than 85%, 90%, 95%, 96%, 97%, 98%, or 99% to a sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 58.

[0054] Sequence identity can be determined in a variety of ways within the scope of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithms employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402, incorporated by reference) is adjusted for searching for sequence similarity. For a discussion of the basic issues in database searches of sequences, see Altschul et al., (1994) NATURE GENETICS 6:119-129, which is fully incorporated by reference. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. Search parameters for histograms, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting a match to a database sequence), cutoffs, matrices and filters are at default settings. The default scoring matrix used by blastp, blastx, tblastn and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, fully incorporated by reference).The four blastn parameters can be adjusted as follows: Q = 10 (gap generation penalty); R = 10 (gap extension penalty); wink = 1 (generating word hits at every wink.sup.th position along the query); and gapw = 16 (setting the window width at which gapped alignments are generated). Equivalent Blastp parameter settings can be Q = 9; R = 2; wink = 1; and gapw = 32. The search can also be performed using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g., -G, cost for open gap [integer]: default = 5 for nucleotides / 11 for proteins; -E, cost for extension gap [integer]: default = 2 for nucleotides / 1 for proteins; -q, penalty for nucleotide mismatch [integer]: default = -3; -r, reward for nucleotide match [integer]: default = 1; -e, expect value [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, drop-off (X) for blast extension in bits: default = 20 for blastn / 7 for others; -X, X drop-off value (in bit) for gapped alignment: default = 15 for all programs but not applicable to blastn; and -Z, final X drop-off value (in bit) for gapped alignment: 50 for blastn, 25 for others). ClustalW for pairwise protein alignment can also be used (default parameters can include, for example, the Blosum62 matrix and Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1).The optimal comparison between sequences available in GCG Package Version 10.0 uses the DNA parameters GAP = 50 (gap generation penalty) and LEN = 3 (gap extension penalty), and the equivalent settings for protein comparison are GAP = 8 and LEN = 2.

[0055] In one aspect, the present invention provides a cytokine-binding protein comprising two fusion proteins, each fusion protein comprising, in the N-terminal to C-terminal direction: a soluble portion of the extracellular domain of a cytokine receptor; an amino acid linker; an immunoglobulin (Ig) hinge region; and an immunoglobulin (Ig) Fc domain, wherein the linker comprises about 5 to about 40 amino acid residues, the two fusion proteins are covalently joined together, and the two extracellular domains together define a binding site for the cytokine.

[0056] The cytokine-binding protein may comprise two of the aforementioned fusion proteins that are covalently joined together, wherein each fusion protein comprises an extracellular domain of a cytokine receptor, and the two extracellular domains together define a binding site for the cytokine. The fusion proteins may be covalently joined, for example, by a disulfide bond between cysteine residues in the Ig hinge region of each fusion protein. In certain embodiments, the fusion protein, which can be either monomeric or multimeric (e.g., dimeric), retains at least 50%, 60%, 70%, 80%, 90% or 95% of the binding activity of the target ligand when compared to the native full-length cytokine receptor.

[0057] In certain embodiments, the cytokine-binding protein of the present invention has a K of 200 nM, 100 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 50 pM, 25 pM or less DIt binds to the cytokine. In certain embodiments, the cytokine-binding protein of the present invention has a K of 200 nM to 100 nM, 200 nM to 20 nM, 200 nM to 10 nM, 200 nM to 5 nM, 200 nM to 1 nM, 200 nM to 50 pM, 200 nM to 25 pM, 100 nM to 20 nM, 100 nM to 10 nM, 100 nM to 5 nM, 100 nM to 1 nM, 100 nM to 50 pM, 100 nM to 25 pM, 20 nM to 10 nM, 20 nM to 5 nM, 20 nM to 1 nM, 20 nM to 50 pM, 20 nM to 25 pM, 10 nM to 5 nM, 10 nM to 1 nM, 10 nM to 50 pM, 10 nM to 25 pM, 5 nM to 1 nM, 5 nM to 50 pM, 5 nM to 25 pM, 1 nM to 50 pM, 1 nM to 25 pM or 50 pM to 25 pM D It binds to the cytokine. In certain embodiments, the cytokine-binding protein of the present invention has a K of 200 nM, 100 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 50 pM, 25 pM or less D It binds to IL-10. In certain embodiments, the cytokine-binding protein of the present invention has a K of 200 nM to 100 nM, 200 nM to 20 nM, 200 nM to 10 nM, 200 nM to 5 nM, 200 nM to 1 nM, 200 nM to 50 pM, 200 nM to 25 pM, 100 nM to 20 nM, 100 nM to 10 nM, 100 nM to 5 nM, 100 nM to 1 nM, 100 nM to 50 pM, 100 nM to 25 pM, 20 nM to 10 nM, 20 nM to 5 nM, 20 nM to 1 nM, 20 nM to 50 pM, 20 nM to 25 pM, 10 nM to 5 nM, 10 nM to 1 nM, 10 nM to 50 pM, 10 nM to 25 pM, 5 nM to 1 nM, 5 nM to 50 pM, 5 nM to 25 pM, 1 nM to 50 pM, 1 nM to 25 pM or 50 pM to 25 pM D It binds to IL-10. K D value can be determined by methods well known in the art such as surface plasmon resonance or bio-layer interferometry method.

[0058] Exemplary fusion proteins of the present invention include proteins comprising amino acid sequences selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 58. For the sake of clarity, Table 1 shows the individual elements of these proteins and the sequences of the individual elements from which the proteins are derived, such as the soluble portion of the extracellular domain of a cytokine receptor, the amino acid linker, the Ig hinge region, and the Ig Fc domain.

[0059]

Table 1

[0060]

Table 2

[0061] II. Preparation of Fusion Proteins Methods for preparing the fusion proteins of the present invention are known in the art. For example, the DNA molecules encoding the disclosed fusion proteins can be chemically synthesized using the sequence information provided herein. The synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, such as expression control sequences, etc., to produce conventional gene expression constructs encoding the desired fusion proteins. The preparation of defined gene constructs is within the scope of conventional techniques in the art. Exemplary nucleic acid sequences encoding the fusion proteins of SEQ ID NOs: 22 - 32, SEQ ID NOs: 37 - 47 can be found in Table 2.

[0062] The nucleic acid encoding the desired fusion protein can be incorporated (ligated) into an expression vector, which can be introduced into a host cell by conventional transfection or transformation techniques. Exemplary host cells are Escherichia coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2) and myeloma cells. The transformed host cells can be grown under conditions that allow the host cell to express the gene encoding the desired fusion protein.

[0063] Specific expression and purification conditions vary depending on the expression system used. For example, when a gene is expressed in E. coli, the gene is first cloned into an expression vector by placing the gene engineered downstream of an appropriate bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. The expressed and secreted protein accumulates in refractile or inclusion bodies and can be recovered after cell disruption by French press or sonication. The inclusion bodies are then solubilized and the protein is refolded and cleaved by methods known in the art.

[0064] When the engineered gene is expressed in a eukaryotic host cell, such as a CHO cell, the gene is first inserted into an expression vector containing an appropriate eukaryotic promoter, a secretion signal, a polyA sequence, and a stop codon, and optionally may contain enhancers and various introns. The gene construct can be introduced into a eukaryotic host cell using conventional techniques.

[0065] Polypeptides containing the disclosed fusion proteins can be produced by growing (culturing) host cells transfected with an expression vector encoding such a protein under conditions that allow expression of the polypeptide. Following expression, the polypeptide can be recovered and purified or isolated using techniques known in the art, such as affinity tags like protein A, protein G, glutathione-S-transferase (GST), or a histidine tag.

[0066] III. Expression Vector The fusion protein of interest can be expressed in the target cells by incorporating the gene encoding the fusion protein of interest into an appropriate expression vector. As used herein, an "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression, and other factors for expression can be supplied by the host cell or in an in vitro expression system. Examples of expression vectors include all those known in the art such as cosmids, plasmids (e.g., naked or contained in liposomes), retrotransposons (e.g., piggyback, sleeping beauty), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate the recombinant polynucleotide of interest.

[0067] In certain aspects, the disclosed expression vectors are viral vectors. The terms "viral vector" and "virus" are used interchangeably herein to refer to either an obligate intracellular parasite that lacks a protein synthesis mechanism or an energy generation mechanism. The viral genome can be RNA or DNA. Viruses useful in the practice of the present invention preferably include recombinant DNA viruses and RNA viruses selected from the families Baculoviridae, Parvoviridae, Picornoviridae, Herpesviridae, Poxyiridae or Adenoviridae that are modified by recombination to encapsidate or not encapsidate. Viruses can be modified by recombinant DNA techniques to include the expression of foreign transgenes and can be engineered to be replication-incompetent, conditionally replicating or replication-competent. Chimeric viral vectors that utilize the advantageous elements of the characteristics of the parental vectors (e.g., Feng et al. (1997) NATURE BIOTECHNOLOGY 15:866-870) can also be useful in the practice of the present invention. It is generally preferred to use a virus derived from the species to be treated, but in some instances it can be advantageous to use vectors from different species that have favorable pathogenic characteristics. For example, equine herpesvirus vectors for human gene therapy are described in PCT Publication WO 98 / 27216. The vector is described as useful for human therapy because equine viruses are not pathogenic to humans. Similarly, ovine adenovirus vectors can be used for human gene therapy because they are claimed to avoid antibodies to human adenovirus vectors. Such vectors are described in PCT Publication WO 97 / 06826.

[0068] In one aspect, the viral vector is an adenovirus. Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. Adenoviruses replicate in the nucleus of mammalian cells using the host's replication machinery. The term "adenovirus" refers to any virus of the genus Adenoviridiae, including but not limited to adenovirus subgroups of human, bovine, ovine, equine, canine, porcine, murine, and simian origin. In particular, human adenoviruses include subgroups A-F and their individual serotypes, and the individual serotypes and subgroups A-F include, but are not limited to, human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Ad11a and Ad11p), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. Vectors derived from human adenovirus types 2 and 5 are preferred. Unless otherwise stated, all adenovirus type 5 nucleotide numbers are related to the NCBI reference sequence AC_000008.1 shown as SEQ ID NO:52 herein.

[0069] The adenovirus replication cycle has two phases: an early phase in which four transcription units (E1, E2, E3, and E4) are expressed, and a late phase that occurs after the initiation of viral DNA synthesis when late transcripts are expressed primarily from the major late promoter (MLP). Late messages encode most of the viral structural proteins. The gene products of E1, E2, and E4 are responsible for transcriptional activation, cell transformation, viral DNA replication, and other viral functions and are required for viral growth.

[0070] The term "operably linked" refers to the linking of polynucleotide elements that are in a functional relationship. A nucleic acid sequence is "operably linked" to another nucleic acid sequence when it is placed in a functional relationship with the other nucleic acid sequence. For example, a promoter or enhancer is operably linked to a gene when it affects the transcription of the gene. Operably linked nucleotide sequences are typically contiguous. However, since enhancers generally function when separated from a promoter by several kilobases and intron sequences can be of variable length, some polynucleotide elements can be operably linked without being directly adjacent and can even function in trans from different alleles or chromosomes.

[0071] IV. Fusion Protein Modification When used as a therapeutic agent, a fusion protein can be optimized (e.g., affinity-matured) to improve biochemical properties such as affinity and / or specificity, to improve biophysical properties such as aggregation, stability, precipitation, and / or non-specific interactions, and / or to reduce immunogenicity. Affinity maturation procedures are within the scope of ordinary skill in the art. For example, diversity can be introduced into the disclosed fusion proteins by DNA shuffling, strand shuffling, CDR shuffling, random mutagenesis, and / or site-directed mutagenesis.

[0072] Generally, an optimized fusion protein has at least the same or substantially the same (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) affinity for a ligand as the non-optimized (or parent) fusion protein from which the optimized fusion protein is derived. Preferably, an optimized fusion protein has a higher affinity for a ligand compared to the parent fusion protein.

[0073] The fusion protein (e.g., the parent and optimized variants) can be engineered to include a particular constant (i.e., Fc) region having a particular effector function (e.g., antibody-dependent cell cytotoxicity (ADCC)). Human constant regions are known in the art.

[0074] Furthermore, if the fusion protein is for use as a therapeutic agent, the fusion protein can be conjugated to an effector agent such as a small molecule toxin or a radionuclide using standard in vitro conjugation chemistry. If the effector agent is a polypeptide, the antibody can be chemically conjugated to the effector or bound to the effector as a fusion protein. The construction of fusion proteins is within the scope of ordinary skill in the art.

[0075] V. Treatment Methods The fusion proteins or expression vectors described above can be used to treat various medical conditions. In certain embodiments, the fusion proteins or expression vectors described above can be used to treat medical conditions mediated by cytokines, such as IL-10. For example, the fusion proteins and expression vectors can be used to treat various cancers or inflammatory diseases.

[0076] As used herein, "treat," "treating," and "treatment" mean the treatment of a disease in a subject, such as a mammal, such as a human. This includes (a) inhibiting the disease, i.e., arresting its development; and (b) alleviating the disease, i.e., causing regression of the diseased state. As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Preferably, such organisms include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), more preferably humans.

[0077] In certain embodiments, the fusion proteins and expression vectors disclosed herein can be used to treat various cancers. The cancer cells are exposed to a therapeutically effective amount of the fusion protein or expression vector so as to inhibit or reduce the growth of the cancer cells. In certain embodiments, administration of a therapeutically effective amount of the fusion protein or expression vector to the cancer cells reduces the IL-10 activity in the cells by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The IL-10 activity can be assayed by Western blot as described in Example 2. In some embodiments, the disclosed fusion proteins or expression vectors can be used to inhibit tumor growth in a subject (e.g., a human patient, also referred to as a human subject), which can be achieved by administering an effective amount of the fusion protein or expression vector to the subject. In certain embodiments, administration of an effective amount of the fusion protein or expression vector to the subject reduces the tumor burden in the subject by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%.

[0078] Examples of cancers include solid tumors, soft tissue tumors, blood tumors, and metastatic lesions. Examples of blood tumors include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), e.g., transformed CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, multiple myeloma, or Richter's syndrome (Richter's Transformation). Examples of solid tumors include malignancies such as sarcomas, adenocarcinomas, and carcinomas of various organ systems, e.g., head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung cancer (NSCLC)), breast, lymphatic system, gastrointestinal (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genital and urogenital tract (e.g., kidney, urothelium, bladder, ovary, uterus, cervix, endometrium, prostate, testis), CNS (e.g., neurons or glial cells, e.g., neuroblastoma or glioma), or skin (e.g., melanoma) that are affected.

[0079] In certain embodiments, the cancer is selected from melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, head and neck cancer, breast cancer, anal cancer, cervical cancer, non-small cell lung cancer, mesothelioma, small cell lung cancer, renal cell carcinoma, prostate cancer, gastroesophageal cancer, colorectal cancer, testicular cancer, bladder cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, cancers of the brain and central nervous system, thyroid cancer, parathyroid cancer (e.g., parathyroid carcinoma), endometrial cancer, neuroendocrine cancer, lymphoma (e.g., Hodgkin's and non-Hodgkin's), leukemia, Merkel cell carcinoma, gastrointestinal stromal tumor, multiple myeloma, uterine cancer, sarcoma, kidney cancer, eye cancer, pancreatic cancer, and germ cell cancer (e.g., ovarian germ cell cancer). In certain embodiments, the cancer can be selected from leukemia, breast cancer, lung cancer, pancreatic cancer, endometrial cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, colorectal cancer, gastric cancer, head and neck cancer, and leukemia. In certain embodiments, the cancer is selected from leukemia, breast cancer, cervical cancer, colorectal cancer, lung cancer, pancreatic cancer, prostate cancer, gastric cancer, head and neck cancer, endometrial cancer, and ovarian cancer.

[0080] In certain embodiments, the fusion protein or expression vector of the present disclosure is administered to a subject in need of a decrease in the level of one or more cytokines (e.g., a subject having an inflammatory condition) to decrease the level of one or more cytokines. In certain embodiments, the disclosed fusion protein or expression vector can be used to treat an inflammatory condition in a subject (e.g., a human subject), which can be achieved by administering to the subject an effective amount of the fusion protein or expression vector.

[0081] As used herein, an inflammatory condition is a disease or condition that wholly or in part features inflammation or an inflammatory response in a patient. Inflammatory conditions treatable using the fusion proteins or expression vectors of the invention can be characterized, for example, based on the main tissue affected, the mechanism of action underlying the condition, or the portion of the immune system that is dysregulated or overly active. In certain embodiments, examples of inflammatory conditions that can be treated include the lungs (e.g., asthma, adult respiratory distress syndrome, bronchitis, pulmonary inflammation, pulmonary fibrosis, and cystic fibrosis), joints (e.g., rheumatoid arthritis, rheumatoid spondylitis, juvenile rheumatoid arthritis, osteoarthritis, gouty arthritis, and other joint conditions), connective tissue, the eyes (e.g., uveitis (including iritis), conjunctivitis, scleritis, and keratoconjunctivitis sicca), the nasal cavity, the intestine (e.g., Crohn's disease, ulcerative colitis, inflammatory bowel disease, inflammatory bowel syndrome, and distal proctitis), the kidneys (e.g., glomerulonephritis, interstitial nephritis, lupus nephritis, nephritis secondary to Wegener's disease, acute renal failure secondary to acute nephritis, Goodpasture's syndrome, post-obstructive syndrome, and tubular ischemia), the liver (e.g., hepatitis (resulting from viral infection, autoimmune response, drug therapy, toxins, environmental factors, or as a secondary consequence of a primary disorder), obesity, biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis), the skin (e.g., psoriasis, eczema, and dermatitis, such as eczematous dermatitis, seborrheic and seborrheic dermatitis, allergic or irritant contact dermatitis, eczema craquelee, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis, radiation dermatitis, and congestive dermatitis), the central nervous system (e.g., multiple sclerosis and neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, or dementia associated with HIV infection), the vasculature (e.g., coronary infarct injury, peripheral vascular disease, myocarditis, vasculitis, angiogenesis in stenosis, atherosclerosis, and vascular diseases associated with type II diabetes), the endocrine system (e.g., autoimmune thyroiditis (Hashimoto's disease), type I diabetes, inflammation in the liver and adipose tissue associated with type II diabetes, and acute and chronic inflammation of the adrenal cortex), the heart, or inflammation of adipose tissue. The present disclosure contemplates that some inflammatory conditions include inflammation in multiple tissues.Furthermore, the present disclosure contemplates that some inflammatory conditions can be classified into multiple categories. In one aspect, the inflammatory condition is an autoimmune disease. Exemplary autoimmune diseases include, but are not limited to, rheumatoid arthritis, psoriasis (including plaque psoriasis), psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, multiple sclerosis, lupus, alopecia, autoimmune pancreatitis, celiac disease, Behçet's disease, Cushing's syndrome, and Graves' disease. In one aspect, the inflammatory condition is a rheumatic-like disorder. Exemplary rheumatic-like disorders include, but are not limited to, rheumatoid arthritis, juvenile arthritis, synovitis, spondylitis, gout, scleroderma, Still's disease and vasculitis. Note that certain categories of conditions overlap. For example, rheumatoid arthritis is an inflammatory rheumatic-like disorder, an inflammatory joint disorder and an autoimmune disorder.

[0082] As used herein, the term "effective amount" refers to the amount of an active ingredient (e.g., the amount of a fusion protein or expression vector of the present invention) sufficient to produce a beneficial or desired result. The effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or route of administration.

[0083] In one aspect, the therapeutically effective amount of the fusion protein is in the range of 0.1 mg / kg to 100 mg / kg, such as 1 mg / kg to 100 mg / kg, 1 mg / kg to 10 mg / kg, 1 mg / kg to 5 mg / kg, 10 mg / kg, 7.5 mg / kg, 5 mg / kg or 2.5 mg / kg. In one aspect, the therapeutically effective amount of an expression vector, such as a recombinant virus, is 10 2 ~10 15 plaque forming units (pfu), such as 10 2 ~10 10 、10 2 ~10 5 、10 5 ~10 15 、10 5 ~10 10 or 10 10 ~10 15It is within the range of plaque-forming units. The dosage depends on variables such as the type and degree of the disease or condition being treated, the overall health of the patient, the in vivo efficacy of the fusion protein or expression vector, the pharmaceutical formulation, and the route of administration. To rapidly achieve the desired blood level or tissue level, the initial dosage can be increased beyond the upper limit. Alternatively, the initial dosage can be smaller than optimal and the daily dosage can be gradually increased during the course of treatment. The human dosage can be optimized in a conventional Phase I dosage escalation study designed to be carried out, for example, from 0.5 mg / kg to 20 mg / kg. The frequency of administration can vary depending on factors such as the route of administration, the dosage, the serum half-life of the antibody, and the disease being treated. Exemplary frequencies of administration are once a day, once a week, and once every two weeks. A preferred route of administration is parenteral, for example, intravenous infusion. The preparation of drugs based on the fusion protein or expression vector is within the scope of ordinary skill in the art. In some embodiments, the fusion protein or expression vector is lyophilized and then reconstituted in buffered saline at the time of administration.

[0084] For therapeutic use, the fusion protein or expression vector is preferably combined with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means a buffer, carrier, and excipient suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, and in a reasonable benefit / risk ratio. The carrier(s) should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, etc., which are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0085] The pharmaceutical composition containing the fusion protein or expression vector disclosed in this specification may exist in unit dosage form and can be prepared by any suitable method. The pharmaceutical composition should be prepared to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, intraocular, intranasal, transdermal, topical, transmucosal, and rectal administration.

[0086] A preferred route of administration for the fusion protein is IV injection. Useful formulations can be prepared by methods known in the pharmaceutical art. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation ingredients suitable for parenteral administration include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffering agents such as acetate, citrate, or phosphate; and agents for adjusting osmotic pressure such as sodium chloride or dextrose.

[0087] Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be protected against microorganisms. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0088] Preferably, the pharmaceutical preparation is sterile. Sterilization can be achieved by any suitable method, such as filtration through a sterile filtration membrane. If the composition is lyophilized, filtration sterilization can be performed before and after lyophilization and reconstitution. In certain embodiments, the delivery vehicle (e.g., recombinant virus) and / or therapeutic agent of the invention is administered in combination with a checkpoint inhibitor, such as an anti-CTLA-4 antibody, an anti-PD-1 antibody or an anti-PD-L1 antibody. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals) and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab (Bavencio®, EMD Serono) and BMS 936559 (Bristol Myers Squibb Co.).

[0089] As used herein, the term administered "in combination" means delivering to a subject two (or more) different treatments such that they overlap at a point in time when there is a therapeutic effect on the subject during the course of suffering from a disorder of the subject. In certain embodiments, there is an overlap in the administration period since delivery of one treatment continues even when delivery of a second treatment has been initiated. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, delivery of one treatment ends before delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective for combination administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with a lesser amount of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In certain embodiments, the delivery is such that reduction of symptoms or other parameters associated with the disorder is greater than that observed when delivering one treatment in the absence of the other treatment. The effects of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that the effect of the first treatment being delivered remains detectable when the second treatment is delivered.

[0090] Throughout the specification, when compositions, devices, and systems are described as having, including, or comprising particular components, or when processes and methods are described as having, including, or comprising particular steps, it is additionally contemplated that there are compositions, devices, and systems of the invention that consist essentially of or consist of the recited components and that there are processes and methods of the invention that consist essentially of or consist of the recited process steps.

[0091] In the present application, when an element or component is said to be included in and / or selected from a list of elements or components described, it should be understood that the element or component can be any one of the elements or components described, or the element or component can be selected from a group consisting of two or more of the elements or components described.

[0092] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether explicit or implicit in the present specification, can be combined in various ways without departing from the spirit and scope of the present invention. For example, when a particular virus is referred to, the virus can be used in various aspects of the compositions of the present invention and / or the methods of the present invention, unless the context indicates otherwise. That is, within the scope of the present application, aspects are described and illustrated in a manner that enables clear and concise application to be described and illustrated, but it is intended and understood that the aspects can be variously combined or separated without departing from the present teachings and the present invention(s). For example, it should be understood that all features described and illustrated herein can be applicable to all aspects of the present invention(s) described and illustrated herein.

[0093] Unless the context and use indicate otherwise, it should be understood that the phrase "at least one of" individually includes each of the things described after the phrase and various combinations of two or more of the things described. The phrase "and / or" with respect to three or more things described should be understood to have the same meaning unless the context indicates otherwise.

[0094] The use of the terms "include", "includes", "including", "have", "has", "having", "contain", "contains" or "containing", including their grammatical equivalents, is generally open-ended and non-limiting and should be understood not to exclude additional elements or steps not recited, unless specifically stated otherwise or understood from the context.

[0095] When the term "about" is used before a quantitative value, the present invention includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a variation of ±10% from the nominal value, unless otherwise indicated or inferred.

[0096] It should be understood that the order of steps or the order of performing certain acts is not important as long as the present invention remains practicable. Further, two or more steps or acts may be performed simultaneously.

[0097] The use of any and all examples or exemplary terms herein, such as "such as" or "including", is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless claimed. Terms herein should not be construed as indicating any unclaimed element as essential to the practice of the present invention.

Examples

[0098] Examples The following examples are merely illustrative and are not intended to limit the scope or content of the present invention in any way.

[0099] Example 1: Construction of IL-10RA Fusion Protein Plasmid This example describes the preparation of plasmids and viral expression vectors encoding an IL-10RA fusion protein.

[0100] A nucleotide sequence encoding a series of human IL-10RA fusion proteins was generated. The first fusion protein, hIL-10R-IgG1 (SEQ ID NO: 58), contained residues 1-229 of human IL-10RA (terminating at SLTRQ), immediately followed by residues 84-330 of the human IgG1 sequence (starting at NVNHK). The second fusion protein, hIL-10R-Fc (SEQ ID NO: 48), contained residues 1-235 of human IL-10RA (terminating at FTVTN), immediately followed by residues 104-324 of human IgG1 (starting at DKTHT). Details of the fusion proteins are shown in Table 3.

[0101]

Table 3

[0102] The nucleotide sequence encoding the fusion protein was cloned into a plasmid for appropriate downstream applications. In particular, recombinant adenoviral vectors that do not express a transgene or express hIL-10R-IgG1 or hIL-10RA-Fc were generated.

[0103] Example 2: IL-10R Fusion Protein Activity A549 cells (human lung cancer cells) were infected with a viral vector that does not express a transgene or expresses hIL-10R-IgG1 or hIL-10RA-Fc, as described in Example 1, at 10 MOI and cultured for 4 days. Conditioned medium was recovered from the cell culture, and THP-1 cells (human leukemic monocytes) were 6Cells were suspended in conditioned medium at cells / ml. The cells were either treated with 50 ng / ml of human IL-10 at 37°C for 30 minutes or kept as a control. To assay for IL-10 activity, cellular proteins extracted from THP-1 cells were examined by Western blot for phosphorylated Stat3. Total Stat3 was used as a loading control.

[0104] IL-10 induced Stat3 phosphorylation in THP-1 cells cultured in conditioned medium derived from cells that did not express the transgene or were infected with a viral vector expressing hIL-10RA-Fc. However, IL-10 did not induce Stat3 phosphorylation in THP-1 cells cultured in conditioned medium derived from cells infected with an hIL10R-IgG expressing virus. These results indicate that the hIL-10R-IgG1 fusion protein blocks IL-10 from activating the Stat3 signaling cascade.

[0105] Incorporation by reference The entire disclosure of each of the patent documents and scientific documents referenced herein is incorporated by reference for all purposes.

[0106] Equivalents The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative rather than restrictive of the invention described herein. Accordingly, the scope of the invention is indicated by the appended claims rather than the foregoing description, and it is intended that all changes which come within the meaning and range of equivalents of the claims are included in the invention.

Claims

1. An isolated fusion protein comprising the amino acid sequence of SEQ ID NO:

58.

2. An IL-10 cytokine binding protein comprising the two fusion proteins according to Claim 1, wherein the two fusion proteins are covalently linked together and the two extracellular domains together define a binding site for binding to the IL-10 cytokine.

3. An isolated nucleic acid comprising the nucleotide sequence encoding the fusion protein according to Claim 1.

4. An expression vector comprising the nucleic acid according to Claim 3.

5. A host cell comprising the expression vector according to Claim 4.

6. (a) culturing the host cell according to Claim 5 under conditions for expressing the fusion protein; and (b) purifying the fusion protein A method for producing a fusion protein, comprising the steps of:

7. A pharmaceutical composition comprising (i) the IL-10 cytokine binding protein according to Claim 2 or the expression vector according to Claim 4; and (ii) at least one pharmaceutically acceptable carrier or diluent.

8. A method for expressing a fusion protein in a target cell, comprising exposing the target cell to an effective amount of the expression vector according to Claim 4 to express the fusion protein.

9. (a) a composition comprising an effective amount of the expression vector according to Claim 4 or (b) the IL-10 cytokine binding protein according to Claim 2, wherein the composition is for use in a method comprising the step of exposing cells, (i) the cells are tumor cells and the method is for inhibiting growth, and the effective amount inhibits the growth of the tumor cells or (ii) the method is for reducing IL-10 activity in the cells, and the effective amount reduces IL-10 activity in the cells.

10. (a) a pharmaceutical composition comprising an effective amount of the expression vector according to Claim 4 or (b) the IL-10 cytokine binding protein according to Claim 2, (i) the pharmaceutical composition is for inhibiting tumor growth in a subject in need thereof, and the effective amount inhibits tumor growth in the subject or (ii) the pharmaceutical composition is for treating cancer in a subject in need thereof, or (iii) The pharmaceutical composition is for treating an inflammatory condition in a subject in need of treatment of the inflammatory condition.

11. The pharmaceutical composition according to claim 10, wherein the cancer is selected from melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, head and neck cancer, breast cancer, anal cancer, cervical cancer, non-small cell lung cancer, mesothelioma, small cell lung cancer, renal cell carcinoma, prostate cancer, gastroesophageal cancer, colorectal cancer, testicular cancer, bladder cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, cancer of the brain and central nervous system, thyroid cancer, parathyroid cancer, endometrial cancer, neuroendocrine cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, Merkel cell carcinoma, gastrointestinal stromal tumor, multiple myeloma, uterine cancer, sarcoma, kidney cancer, eye cancer, pancreatic cancer and germ cell cancer.

12. The pharmaceutical composition according to claim 10 or 11, wherein the subject is human.

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