IL10 inverse monomer

Abstract

This disclosure relates to engineered polypeptides containing components of the IL-10 molecule that modulate IL-10 related signaling, and to methods of using the same. In some aspects, the polypeptides of this disclosure exhibit cell type-biased activity. In some aspects, the polypeptides of this disclosure substantially retain the anti-inflammatory properties of IL-10 but significantly reduce the pro-inflammatory properties of IL-10. This disclosure provides methods of using the engineered polypeptides in the treatment of diseases. TIFF2026519565000106.tif214158

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 505,651, filed on 1 June 2023, the disclosure of which is incorporated herein by reference in its entirety for any purpose. [Background technology]

[0002] background Cytokines and growth factor ligands typically signal through the multimerization of cell surface receptor subunits. In some cases, cytokines act as multispecific (e.g., bispecific or triplicate) ligands, promoting the association of the extracellular domains of receptor subunits, thereby bringing the intracellular domains of the receptor subunits into proximity and facilitating intracellular signaling. The properties of cytokine ligands and their interaction with the extracellular domains of receptor subunits determine which receptor subunits associate and how to form ligand-receptor complexes, and the intracellular signaling characteristic of such complexes.

[0003] The intracellular domains of several cytokine receptor subunits contain a Janus kinase ("JAK") binding domain. The JAK binding domain is typically located in the box1 / box region of the intracellular domain of the cytokine receptor subunit near the inner surface of the cell membrane. Intracellular Janus kinase associates with the JAK binding domain and phosphorylates it. In mammalian cells, four Janus kinases have been identified: JAK1, JAK2, JAK3, and TYK2. Ihle, et al. (1995) Nature 377(6550):591-4, 1995 (Non-patent Literature 1); O'Shea and Plenge (2012) Immunity 36(4):542-50 (Non-patent Literature 2). When the intracellular domains of cytokine receptor subunits containing the JAK binding domain are brought into close proximity, Janus kinase promotes transphosphorylation of the JAK binding domain. Phosphorylation of JAK induces a conformational change in JAK, resulting in JAK's ability to further phosphorylate other intracellular proteins. The resulting phosphorylation cascade leads to the activation of multiple intracellular factors, thereby transmitting intracellular signals related to cytokine ligand-mediated receptor activation. In some cases, the intracellular proteins phosphorylated by JAK are members of the signaling transcription factor ("STAT") protein family. Seven members of the mammalian STAT family, namely STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6, have been identified to date. (Delgoffe, et al., (2011) Curr Opin Immunol. 23(5):632-8 (Non-patent Literature 3); Levy and Darnell (2002) Nat Rev Mol Cell Biol. 3(9):651-62 (Non-patent Literature 4); and Murray, (2007) J Immunol. 178(5):2623-9 (Non-patent Literature 5). The selective interaction of activated JAK and STAT proteins, collectively referred to as the JAK / STAT pathway, is involved in a wide variety of intracellular responses observed in response to cytokine binding.Such intracellular responses, initiated by the binding of cytokines to their receptors, are often referred to as downstream signaling.

[0004] The cytokine human interleukin-10 (hIL10), also known as a human cytokine synthesis inhibitor (CSIF), is classified into a group of cytokines including type (class)-2 cytokines, IL19, IL20, IL22, IL24 (Mda-7), and IL26, interferons (IFN-α, -β, -γ, -δ, -ε, -κ, -Ω, and -τ), and interferon-like molecules (such as limitin, IL28A, IL28B, and IL29). hIL10 is a non-covalent homodimer composed of two hIL10 monomer polypeptides. Each hIL10 monomer polypeptide is a 160-amino acid polypeptide with two intramolecular disulfide bonds. Each hIL10 monomer is expressed as a proprotein consisting of 178 amino acids, the first 18 of which contain a signal peptide. hIL10 is primarily expressed by macrophages, but its expression has also been detected in activated T cells, B cells, mast cells, NK cells, dendritic cells, eosinophils, neutrophils, and monocytes.

[0005] Human IL-10 exerts its effects on cells through its interaction with the hIL-10 receptor (hIL-10R). hIL-10R is a type II cytokine receptor comprising the hIL-10Rα subunit and the hIL-10Rβ subunit, also known as hIL-10R1 and hIL-10R2, respectively. The hIL-10Rα receptor subunit is a “private” or “exclusive” subunit, restricted to the hIL-10 receptor alone. In contrast, the hIL-10Rβ subunit is shared with other cytokine receptors, including the receptor complexes for IL-22, IL-26, IL-28, and interferon-lambda L1 (IFNλ1). Activation of hIL-10R is characterized by the binding of each hIL-10 monomer to one hIL-10Rα subunit and one hIL-10Rβ subunit of hIL-10R. Each monomer of the dimeric hIL10 cytokine associates with one hIL10Rα subunit and one hIL10Rβ subunit to form a hexameric ligand / receptor hIL10R complex consisting of two hIL10 monomers, two hIL10Rα subunits, and two hIL10Rβ subunits.

[0006] The hIL10Rα receptor subunit is a transmembrane protein expressed as a 578-amino acid proprotein containing a 21-amino acid signal sequence at the N-terminus. The amino acid sequence of the mature canonical hIL10Ra receptor subunit is as follows: This is a 557-amino acid polypeptide with the codename TIFF2026519565000002.tif78128 (UniProt reference number Q13651). Residues 22-235 of SEQ ID NO:24 (amino acids 1-214 of the mature hIL10Rβ protein) correspond to the extracellular domain (ECD), residues 236-256 of SEQ ID NO:24 (amino acids 215-235 of the mature hIL10Rβ protein) correspond to the transmembrane domain (TM), and residues 257-578 of SEQ ID NO:24 (amino acids 236-557 of the mature hIL10Rβ protein) correspond to the intracellular domain (ICD).

[0007] The human IL10Rβ (hIL10Rβ) receptor subunit is a transmembrane protein expressed as a 325-amino acid proprotein containing a 19-amino acid N-terminal signal. The amino acid sequence of the mature canonical hIL10Rβ receptor subunit is as follows: This is a 306-amino acid polypeptide, TIFF2026519565000003.tif44128 (UniProt reference number Q08334). Amino acids 20-220 (amino acids 1-201 of the mature hIL10Rβ protein) correspond to the extracellular domain, amino acids 221-242 (amino acids 202-223 of the mature hIL10Rβ protein) correspond to the 22-amino acid transmembrane domain, and amino acids 243-325 (amino acids 224-306 of the mature hIL10Rβ protein) correspond to the intracellular domain.

[0008] The mouse IL10Rβ (mIL10Rβ) receptor subunit is expressed as a 349-amino acid proprotein containing a 19-amino acid N-terminal signal sequence. The amino acid sequence of the mature canonical hIL10Rb receptor subunit is as follows: This is a 330-amino acid polypeptide, TIFF2026519565000004.tif44128.

[0009] mIL10Rβ (mIL10R2) is referenced as entry Q61190 in the UniProtKB database. Amino acids 20-220 (amino acids 1-201 in the mature protein) correspond to the extracellular domain, amino acids 221-241 (amino acids 202-222 in the mature protein) correspond to the 21-amino acid transmembrane domain, and amino acids 242-349 (amino acids 223-330 in the mature protein) correspond to the intracellular domain.

[0010] The interactions between IL10 and its receptor and receptor subunits have been studied and documented in the scientific literature. Pletnev et al. have provided information on the structure of the soluble receptor chain of IL10Rβ and the ternary complex of IL10 / sIL10Rα / sIL10Rβ, as well as the residues involved in ligand-receptor and receptor-receptor interactions. Pletnev, et al. (2005) BMC Structural Biology 5:10 (Non-patent Literature 6). The interaction between hIL10 and the hIL10Rα receptor subunit is a specific high-affinity interaction, while the association between hIL10 and hIL10Rβ is a relatively low-affinity interaction. Reports suggest that the interaction between hIL10 and hIL10Rα induces conformational changes in hIL10 and / or hIL10Rα, thereby promoting the binding of the [hIL10:hIL10Rα] complex to hIL10Rβ. The formation of the ternary [hIL10:hIL10Rα:hIL10Rβ] complex has been suggested to be a limiting factor in initiating hIL10 signaling.

[0011] The interaction between IL-10 and IL10R leads to the activation of JAK1 (associated with hIL10Rα) and Tyk2 (associated with hIL10Rβ), inducing the activation of STAT1, STAT3, and in some cells, STAT5. STAT3 is directly recruited to the hIL-10 / hIL-10R complex via either of two tyrosine residues in the hIL10Rα cytoplasmic domain, which are phosphorylated in response to hIL-10 and required for hIL-10 signaling. Homodimerization of STAT3 leads to its release from the receptor and the translocation of phosphorylated STAT3 homodimers into the nucleus, where the phosphorylated STAT3 homodimers bind to STAT3-binding elements in the promoters of numerous genes, including the IL10 promoter, which are positively regulated by STAT3. The intracellular domain of the hIL10 receptor possesses sequences associated with its anti-inflammatory activity that are not shared by other STAT3-activated cytokine receptors. Riley, et al. (1999) Journal of Biological Chemistry 274(23):15967-16664 (Non-Patent Document 7).

[0012] The expression of hIL10Rα receptor subunits and IL10Rb receptor subunits varies with respect to cell type and cellular activation state. The activation state of cells expressing hIL10Rα can lead to substantial fluctuations in expression levels. In hematopoietic cells that constitutively express low levels of hIL10Rα, hIL10Rα expression is often substantially upregulated by various stimuli. In contrast to hIL10Rα, which is mainly expressed on hematopoietic cells, the IL10Rb receptor subunit is ubiquitous. Certain cell types express hIL10Rβ at varying levels, but the level of hIL10Rβ expression in a given cell type is typically less affected by cellular activation state than hIL10Rα.

[0013] hIL10 is associated with a wide range of functions and exhibits both immunosuppressive and immunostimulatory activity through its interactions with T cells, B cells, macrophages, and antigen-presenting cells (APCs). The immunosuppressive activity of hIL10 is well-established. hIL10 is associated with the suppression of IL1α, IL1β, IL6, IL8, TNFα, GM-CSF, and G-CSF expression in activated monocytes and activated macrophages, as well as the suppression of pro-inflammatory cytokine interferon-gamma (INFγ) production by NK cells. However, hIL10 also exhibits an immunostimulatory effect by stimulating the production of pro-inflammatory cytokine IFN-γ by CD8+ T cells. The immunostimulatory and immunosuppressive properties of hIL10 have proven to be a challenge in the clinical application of hIL10 in the treatment of human diseases.

[0014] Saxton et al. have described the interaction between IL10 and the IL10Rb subunit and amino acid residues involved in the binding of IL10 to IL10Rβ, and that modifications of such residues may potentially result in IL10 variants that retain the immunosuppressive function of IL10 on myeloid cells but have reduced pro-inflammatory activity on CD8+ T cells. Saxton, et al. (2021) Science 371(6535), eabc8433 (Non-patent Literature 8). [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Ihle,et al.(1995)Nature 377(6550):591-4,1995 [Non-Patent Document 2] O'Shea and Plenge (2012) Immunity 36(4):542-50 [Non-Patent Document 3] Delgoffe,et al.,(2011)Curr Opin Immunol.23(5):632-8 [Non-Patent Document 4] Levy and Darnell (2002) Nat Rev Mol Cell Biol.3(9):651-62 [Non-Patent Document 5] Murray,(2007)J Immunol.178(5):2623-9 [Non-Patent Document 6] Pletnev, et al. (2005) BMC Structural Biology 5:10 [Non-Patent Document 7] Riley, et al. (1999) Journal of Biological Chemistry 274(23):15967-16664 [Non-Patent Document 8] Saxton,et al.(2021)Science 371(6535),eabc8433 [Overview of the project]

[0016] Summary of this disclosure This disclosure provides compositions useful for modulating interleukin-10 (IL10)-mediated signaling. In particular, this disclosure provides inverted monomers and dimers thereof that are partial agonists of STAT3-mediated signaling ("STAT3 signaling"). In some embodiments, the inverted monomers and dimers described herein activate STAT3 signaling in some cell types and degrade STAT3 signaling in other cell types. In some embodiments, the inverted monomers and dimers described herein activate STAT3 signaling in myeloid cells and degrade STAT3 signaling in lymphocytes. In some embodiments, the inverted monomers and dimers described herein showed preferential activation of STAT3 signaling in myeloid cells compared to lymphocytes compared to with hIL10.

[0017] In one aspect, a polypeptide containing the amino acid sequence of formula 1 is provided: [A]-L1x-[B]-L2-[C]-L3-[D]-L4y-[E]-L5-[F](1) During the ceremony, x and y are chosen independently of 0 (non-existent) or 1 (existent). [A] is the amino acid sequence Compared to TIFF2026519565000005.tif4128, this polypeptide has 0, 1 or 2 amino acid substitutions. x=0 (no linker, L1 does not exist), or x=1, and L1 contains a polypeptide linker of 1-5 amino acids. [B] is the amino acid sequence Compared to TIFF2026519565000006.tif4128, this polypeptide has 0, 1, or 2 amino acid substitutions. L2 contains a linker of 10-25 amino acids. [C] represents the amino acid sequence Compared to TIFF2026519565000007.tif4128, this polypeptide has 0, 1, or 2 amino acid substitutions. L3 contains a linker of 4-11 amino acids. [D] is the amino acid sequence Compared to TIFF2026519565000008.tif4128, this polypeptide has 0, 1 or 2 amino acid substitutions. y=0 (L4 does not exist), or y=1, and L4 contains a polypeptide linker of 1-5 amino acids. [E] is the amino acid sequence Compared to TIFF2026519565000009.tif4128, this polypeptide has 0, 1, or 2 amino acid substitutions. L5 contains a linker of 1 to 7 amino acids, and [F] represents the amino acid sequence This polypeptide has 0, 1, or 2 amino acid substitutions compared to TIFF2026519565000010.tif4128.

[0018] In some embodiments, L1, L2, L3, L4 and / or L5 include a GS-linker. In some embodiments, L1 is the amino acid glutamine (Q). In some embodiments, L2 is the amino acid sequence The polypeptide has TIFF2026519565000011.tif4158. In some embodiments, L3 is a polypeptide having the amino acid sequence QLDNLLL (SEQ ID NO: 8). In some embodiments, L4 is the amino acid glycine ("G") or contains the amino acid sequence GY. In some embodiments, L5 is a polypeptide having the amino acid sequence QDPD (SEQ ID NO: 9). In some embodiments, the polypeptide contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11.

[0019] In some embodiments, the polypeptide is an amino acid sequence Includes TIFF2026519565000012.tif24139, or In some embodiments, the polypeptide is an amino acid sequence It contains an amino acid sequence that has at least 95% sequence identity with TIFF2026519565000013.tif24139, or SEQ ID NO:10 or SEQ ID NO:11.

[0020] In some embodiments, the polypeptide contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to an hIL10 reverse monomer selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.

[0021] In some embodiments, polypeptides comprising formula (2) are provided: [Monomer 1]-Linker x -[monomer 2](2) In the formula, monomer 1 and monomer 2 are polypeptides of formula 1, and monomer 1 and monomer 2 are either the same or different, and x = 0 (no linker) or 1 (a linker exists).

[0022] In some embodiments, the polypeptide of formula (2) is arranged in the order from amino to carboxyl, (a) Below: (i) A first polypeptide having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), 117-159 (SEQ ID NO: 13), and 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature wild-type human IL-10 (SEQ ID NO: 20), (ii) The first linker where x = 0 (no linker exists) or 1 (a linker exists) x and, (iii) Numbered according to mature wild-type hIL10 (SEQ ID NO:20), amino acid residues 1-160 of human IL-10 (SEQ ID NO:20), amino acid residues 2-160 of human IL-10 (SEQ ID NO:36), amino acid residues 3-160 of human IL-10 (SEQ ID NO:37), amino acid residues 4-160 of human IL-10 (SEQ ID NO:38), amino acid residues 5-160 of human IL-10 (SEQ ID NO:39), amino acid residues 6-160 of human IL-10 (SEQ ID NO:40), amino acid residues 7-160 of human IL-10 (SEQ ID NO:41), amino acid residues 8-160 of human IL-10 (SEQ ID NO:42), amino acid residues 9-160 of human IL-10 (SEQ ID NO: A second polypeptide containing amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 10-160 of human IL-10 (SEQ ID NO:44) and amino acid residues 11-160 of human IL-10 (SEQ ID NO:45), and Includes monomer 1, (b) A second linker where y = 0 (no linker exists) or 1 (a linker exists). y , and (c) Numbered according to mature human IL-10 (SEQ ID NO:20), amino acid residues 1-116 of human IL-10 (SEQ ID NO:15), amino acid residues 2-116 of human IL-10 (SEQ ID NO:16), amino acid residues 3-116 of human IL-10 (SEQ ID NO:17), amino acid residues 4-116 of human IL-10 (SEQ ID NO:18), amino acid residues 5-116 of human IL-10 (SEQ ID NO:19), amino acid residues 6-116 of human IL-10 (SEQ ID NO:29), amino acid residues 7-116 of human IL-10 (SEQ ID NO:30), amino acid residues 8-116 of human IL-10 (SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID Monomer 2) containing a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 10-116 of human IL-10 (SEQ ID NO:33) and amino acid residues 11-116 of human IL-10 (SEQ ID NO:34). Includes.

[0023] In some embodiments, the polypeptide of formula (2) is arranged in the order from amino to carboxyl, (a) Below: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), 117-159 (SEQ ID NO: 13), and 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature human IL-10 (SEQ ID NO: 20), (ii) First linker x , (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 (SEQ ID NO: 15) of human IL-10. Includes monomer 1, (b) Second linkery , (c) Below: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), 117-159 (SEQ ID NO: 13), and 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature human IL-10 (SEQ ID NO: 20), (ii) Third linker z and, (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1 to 116 (SEQ ID NO: 15) of human IL-10, Includes monomer 2 Includes.

[0024] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity hIL10 reverse monomers selected from the group consisting of SEQ ID NO: 46-51.

[0025] In some embodiments, the polypeptide exhibits cell type biased activity compared to wild-type IL10 derived from the inverse IL10 monomer. In some embodiments, the polypeptide exhibits (a) a marked level of at least one anti-inflammatory property of wild-type IL10 and (b) a markedly reduced level of at least one pro-inflammatory property of wild-type IL10. In some embodiments, at least one anti-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IL1β, TNFα, or IL6 in bone marrow cells. In some embodiments, at least one pro-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IFNγ, granzyme A, or granzyme B in T cells. In some embodiments, at least one anti-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IL1β, TNFα, or IL6 in bone marrow cells, and at least one pro-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IFNγ, granzyme A, or granzyme B in T cells.

[0026] In some embodiments, cell type biasing activity is phospho-STAT3 production. In some embodiments, the pSTAT3 Emax of polypeptides is greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70% of the pSTAT3 Emax of wild-type hIL10 in bone marrow cells.

[0027] In some embodiments, the pSTAT3 Emax in lymphocytes is less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of the pSTAT3 Emax of wild-type hIL10 in lymphocytes.

[0028] In some embodiments, (a) the pSTAT3 Emax of the polypeptide is greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70% of the pSTAT3 Emax of wild-type hIL10 in bone marrow cells, and (b) the pSTAT3 Emax in lymphocytes is less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of the pSTAT3 Emax of wild-type hIL10 in lymphocytes.

[0029] In some embodiments, polypeptides comprising formula (2) are provided: [Monomer 1]-Linker x -[monomer 2](2) In the formula, monomer 1 and monomer 2 each independently contain polypeptides selected from the polypeptides described in claim 1, and x = 0 (no linker present) or 1 (a linker present).

[0030] In some embodiments, monomer 1 and monomer 2 each independently contain polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.

[0031] In some embodiments, monomer 1 and monomer 2 each contain a polypeptide having the amino acid sequence of SEQ ID NO:10.

[0032] In some embodiments, monomer 1 and monomer 2 each contain a polypeptide having the amino acid sequence of SEQ ID NO:11.

[0033] In some embodiments, x is 1 (a linker exists), and the linker includes a GS-linker.

[0034] In some embodiments, x is 0 (the linker does not exist).

[0035] In some embodiments, the polypeptide of formula (2) is arranged in the order from amino to carboxyl, (a) Below: (i) A first polypeptide having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), 117-159 (SEQ ID NO: 13), and 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature wild-type human IL-10 (SEQ ID NO: 20), (ii) The first linker where x = 0 (no linker exists) or 1 (a linker exists) x and, (iii) A second polypeptide comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide sequence selected from the group consisting of amino acid residues 1-160 (SEQ ID NO:20), amino acid residues 2-160 (SEQ ID NO:36), amino acid residues 3-160 (SEQ ID NO:37), amino acid residues 4-160 (SEQ ID NO:38), amino acid residues 5-160 (SEQ ID NO:39), amino acid residues 6-160 (SEQ ID NO:40), amino acid residues 7-160 (SEQ ID NO:41), amino acid residues 8-160 (SEQ ID NO:42), amino acid residues 9-160 (SEQ ID NO:43), amino acid residues 10-160 (SEQ ID NO:44), and amino acid residues 11-160 (SEQ ID NO:45) of human IL-10 numbered according to mature wild-type hIL10 (SEQ ID NO:20) and comprising Monomer 1, (b) A second linker where y = 0 (no linker present) or 1 (linker present), y and (c) Numbered according to mature human IL-10 (SEQ ID NO:20), amino acid residues 1-116 of human IL-10 (SEQ ID NO:15), amino acid residues 2-116 of human IL-10 (SEQ ID NO:16), amino acid residues 3-116 of human IL-10 (SEQ ID NO:17), amino acid residues 4-116 of human IL-10 (SEQ ID NO:18), amino acid residues 5-116 of human IL-10 (SEQ ID NO:19), amino acid residues 6-116 of human IL-10 (SEQ ID NO:29), amino acid residues 7-116 of human IL-10 (SEQ ID NO:30), amino acid residues 8-116 of human IL-10 (SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID Monomer 2) containing a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 10-116 of human IL-10 (SEQ ID NO:33) and amino acid residues 11-116 of human IL-10 (SEQ ID NO:34). Includes.

[0036] In some embodiments, the polypeptide of formula (2) is arranged in the order from amino to carboxyl, (a) Below: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), 117-159 (SEQ ID NO: 13), and 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature human IL-10 (SEQ ID NO: 20), (ii) The first linker where x = 0 (no linker exists) or 1 (a linker exists) x , (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 (SEQ ID NO: 15) of human IL-10. Includes monomer 1, (b) A second linker where y = 0 (no linker exists) or 1 (a linker exists). y , (c) Below: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), 117-159 (SEQ ID NO: 13), and 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature human IL-10 (SEQ ID NO: 20), (ii) A third linker where z = 0 (no linker exists) or 1 (a linker exists) z and, (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1 to 116 (SEQ ID NO: 15) of human IL-10, Includes monomer 2 Includes.

[0037] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity hIL10 reverse monomers selected from the group consisting of SEQ ID NO: 46-51.

[0038] In some embodiments, polypeptides are PEGylated. In some embodiments, the PEG molecule is linear or branched and has a molecular weight of about 10 kD to about 80 kD. In some embodiments, the PEG molecule is a 40 kD branched PEG molecule containing two 20 kD arms. In some embodiments, the PEG molecule is covalently bonded to the N-terminus of the polypeptide.

[0039] In some embodiments, the polypeptide exhibits cell-type biased activity compared to wild-type IL10 derived from the reverse IL10 monomer. In some embodiments, the polypeptide exhibits (a) a marked level of at least one anti-inflammatory property of wild-type IL10 and (b) a markedly reduced level of at least one pro-inflammatory property of wild-type IL10. In some embodiments, at least one anti-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IL1b, TNFa, or IL6 in bone marrow cells. In some embodiments, at least one pro-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IFNγ, granzyme A, or granzyme B in T cells. In some embodiments, at least one anti-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IL1b, TNFa, or IL6 in bone marrow cells, and at least one pro-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IFNγ, granzyme A, or granzyme B in T cells.

[0040] In some embodiments, cell type bias activity is phospho-STAT3 production. In some embodiments, the pSTAT3 Emax of polypeptides is greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70% of the pSTAT3 Emax of wild-type hIL10 in myeloid cells. In some embodiments, the pSTAT3 Emax in lymphocytes is less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of the pSTAT3 Emax of wild-type hIL10 in lymphocytes. In some embodiments, (a) the pSTAT3 Emax of the polypeptide is greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70% of the pSTAT3 Emax of wild-type hIL10 in bone marrow cells, and (b) the pSTAT3 Emax in lymphocytes is less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of the pSTAT3 Emax of wild-type hIL10 in lymphocytes.

[0041] Nucleic acid sequences encoding polypeptides described above or elsewhere in this specification are also provided.

[0042] Recombinant vectors are also provided, comprising nucleic acid sequences described above or elsewhere in this specification, functionally ligated to one or more expression control sequences.

[0043] Recombinant cells transformed with the recombinant vectors described above or elsewhere in this specification are also provided.

[0044] A method for producing the polypeptide described above or elsewhere in this specification is also provided, comprising the steps of (a) culturing the host cells described above or elsewhere in this specification under conditions suitable for polypeptide expression, and (b) recovering the polypeptide from the host cell culture. In some embodiments, the host cells are mammalian host cells. In some embodiments, the host cells are bacterial cells.

[0045] Compositions are also provided comprising a polypeptide as described above or elsewhere in this specification, a nucleic acid sequence as described above or elsewhere in this specification, or a recombinant vector as described above or elsewhere in this specification, and one or more pharmaceutically acceptable salts, excipients and / or diluents.

[0046] Methods for treating a mammalian subject suffering from a disease, disorder, or condition are also provided, comprising the step of administering a therapeutically effective amount of a polypeptide or composition described above or elsewhere in this specification to the subject. In some embodiments, the disease, disorder, or condition is an autoimmune disease, disorder, or condition. In some aspects, autoimmune diseases, disorders, or conditions include ulcerative colitis, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes including type 1 or type 2 diabetes, inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, and SEA syndrome (Seronegativity Enthesopathy Arthropathy). The treatment is selected from the group consisting of juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarthritis, polyarthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteritis arthritis, reactive arthritis, Reiter's syndrome, and SEA syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome). In some embodiments, the disease, disorder, or condition is cancer associated with chronic inflammation. In some embodiments, the treatment prevents the progression of the disease, disorder, or condition. In some embodiments, the treatment improves one or more symptoms of the disease, disorder, or condition.

[0047] A method for preventing a disease, disorder or condition in a mammal at risk of developing a disease, disorder or condition is also provided, comprising the step of administering a preventive effective amount of the composition described above or elsewhere in this specification to the subject before the onset of symptoms of the disease, disorder or condition described above or elsewhere in this specification, wherein the disease, disorder or condition is cancer associated with chronic inflammation. [Brief explanation of the drawing]

[0048] [Figure 1] Figure 1 shows thermal stability data for the IL10 inverse monomer of this disclosure, as will be described in more detail in the examples. The data shows that the hIL10 inverse monomer has enhanced thermal stability compared to hIL10. [Figure 2A] Figure 2A shows the results of a flow cytometry assay evaluating intracellular STAT3 levels (y axis) in human monocytes exposed to various concentrations (x axis) of wild-type human IL10 and inverse hIL10 monomers, as described in more detail in the examples. [Figure 2B] Figure 2B shows the results of a flow cytometry assay evaluating intracellular STAT3 levels (y axis) in human naive CD8 T cells exposed to various concentrations (x axis) of wild-type human IL10 and inverse hIL10 monomers, as described in more detail in the examples. [Figure 3] Figure 3 shows the results of a human monocyte activity assay to detect IL-1β secretion (y axis) in human monocytes exposed to various concentrations (x axis) of wild-type hIL10 (h_SM0043_AA) and the inverse monomer of the present disclosure (h_DR1061_AA), as will be described in more detail in the Examples. [Figure 4A] Figures 4A to 4C show the secretion levels (y axis) of IFNγ (Figure 4A), granzyme A (Figure 4B), and granzyme B (Figure 4C) in activated human CD8+ T cells in response to various concentrations (x axis) of wild-type hIL10 (h_SM0043_AA) and the inverse monomer of this disclosure (h_DR1061_AA), as will be described in more detail in the examples. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 5A] Figure 5A shows the results of a flow cytometry assay evaluating intracellular STAT3 levels (y axis) in mouse bone marrow cells exposed to various concentrations (x axis) of wild-type mouse IL10 (m_DR756_AA) and reverse mMIL10 monomer (m_WC161_AA), as described in more detail in the examples. [Figure 5B] Figure 5B shows the results of a flow cytometry assay evaluating intracellular STAT3 levels (y axis) in mouse CD8 T cells exposed to various concentrations (x axis) of wild-type mouse IL10 (m_DR756_AA) and reverse mMIL10 monomer (m_WC161_AA), as described in more detail in the examples. [Figure 6A] Figure 6A shows the results of experiments to evaluate IL6 secretion (y axis) in mouse splenocytes in response to various concentrations (x axis) of wild-type mouse IL10 (m_DR756_AA) and inverse mMIL10 monomer (m_WC161_AA), as described in more detail in the examples. [Figure 6B] Figure 6B shows the results of experiments to evaluate TNFα secretion (y axis) in mouse splenocytes in response to various concentrations (x axis) of wild-type mouse IL10 (m_DR756_AA) and reverse mMIL10 monomer (m_WC161_AA), as described in more detail in the Example Mouse Splenocyte TNF-Alpha Assay. [Figure 7] Figure 7 shows the results of experiments to evaluate granzyme B (y axis) produced in activated mouse CD8+ T cells in response to various concentrations (x axis) of wild-type mouse IL10 (m_DR756_AA) and inverse mRNA (m_WC161_AA), as described in more detail in the examples. [Figure 8]Figure 8 shows experimental results on the survival (y axis) of activated mouse CD8+ T cells in response to various concentrations (x axis) of wild-type mouse IL10 (m_DR756_AA) and inverse mRNA (m_WC161_AA), as described in more detail in the examples. [Figure 9] Figure 9 shows the results of pharmacokinetic studies to evaluate the time-dependent (x-axis) serum stability (y-axis) of PEGylated mouse reverse IL10 monomer (WC161) at various dose levels compared to PEGylated wild-type mouse IL10 control (DR756), as described in more detail in the Examples. [Figure 10] Figure 10 shows the results of experiments to evaluate the level of cell surface expression (y axis) of CD64 on human monocytes treated with various concentrations (x axis) of IL10 protein (0.1 pM to 100 nM) over 48 hours at 37°C, as described in more detail in the Examples. [Figure 11] Figure 11 shows the alignment of the human inverse monomer (DR1060_aa / 1-171; SEQ ID NO:27) and mouse inverse monomer (WC161_aa / 1-172; SEQ ID NO:28) of this disclosure. [Figure 12] Figure 12A shows the results of a flow cytometry assay evaluating intracellular STAT3 levels (y axis) in human monocytes exposed to various concentrations (x axis) of wild-type human IL10 (h_DR757_AA) and inverse hIL10 monomers (h_DR1060_AA and h_DR1061_AA), as described in more detail in the examples. Figure 12B shows the results of a flow cytometry assay evaluating intracellular STAT3 levels (y axis) in human naive CD8 T cells exposed to various concentrations (x axis) of wild-type human IL10 (h_DR757_AA) and inverse hIL10 monomers (h_DR1060_AA and h_DR1061_AA), as described in more detail in the examples. [Figure 13]Figure 13 shows data on mouse body weight over time (x-axis) in a DSS model of ulcerative colitis, as described in more detail in the examples. The upper panel reflects simple body weight measurements, while the lower panel provides animal body weight compared to initial body weight at the start of the study. [Figure 14] Figure 14 shows data on body weight (y axis) of mice treated with the test substance (x axis) in a DSS model of ulcerative colitis, as evaluated at the end of the experiment (D15), as described in more detail in the examples. [Figure 15] Figure 15 shows data on colon length (y axis) of mice treated with the test substance (x axis) in a DSS model of ulcerative colitis, as evaluated at the end of the experiment (D15), as described in more detail in the examples. [Figure 16] Figure 16 shows data on hematocrit levels (y axis) from mice treated with the test substance (x axis) in a DSS model of ulcerative colitis, as evaluated on day 4 (D4) of the experiment, as described in more detail in the examples. [Figure 17] Figure 17 shows data on the percentage (y axis) of peritoneal CD163+ macrophages (CD163+) derived from mice treated with the test agent (x axis) in a DSS model of ulcerative colitis, as will be described in more detail in the examples. [Figure 18] Figure 18 shows data on the percentage (y axis) of peritoneal CD64 macrophages (OCT) from mice treated with the test agent (x axis) in a DSS model of ulcerative colitis. [Figure 19-1] Figure 19 shows time-dependent (x-axis) serum cytokine levels (y-axis) from mice for various treatments in a DSS model of ulcerative colitis, as described in more detail in the examples. [Figure 19-2] See the explanation in Figure 19-1. [Figure 19-3] See the explanation in Figure 19-1. [Figure 20-1]Figure 20 shows time-dependent (x-axis) serum cytokine levels (y-axis) from mice for various treatments in a DSS model of ulcerative colitis, as described in more detail in the examples. [Figure 20-2] See the explanation in Figure 20-1. [Figure 21] Figure 21 shows data on the percentage of epithelial damage (y axis) from mice treated with the test substance (x axis) as measured on D15 of a DSS model of ulcerative colitis, as described in more detail in the examples. [Figure 22] Figure 22, left panel shows data on the level of CD11b cells (y axis) in the intestinal mucosa of mice treated with the test agent (x axis) as measured at D15 of the DSS model of ulcerative colitis, as described in more detail in the examples. Figure 22, right panel shows data on the level of Th17 cells (y axis) in the intestinal mucosa of mice treated with the test agent (x axis) as measured at D15 of the DSS model of ulcerative colitis, as described in more detail in the examples. [Figure 23] Figure 23 shows the results of pharmacokinetic studies conducted in mice to evaluate the serum concentration of the test activator over time (x-axis) in response to administration of various levels of the test activator, as described in more detail in the examples. [Figure 24] Figure 24 shows data on the level of STAT3 induction (y-axis) in CD4 T cells (top panel), CD8 T cells (center panel), and B cells (bottom panel) in response to administration of various levels of test activators, as will be described in more detail in the examples. [Modes for carrying out the invention]

[0049] Detailed explanation To facilitate understanding of this disclosure, certain terms and phrases are defined below and throughout this specification. The definitions provided herein are not limiting and should be read with the knowledge of those skilled in the art in mind.

[0050] Before describing the methods and compositions described herein, it should be understood that the present invention is not limited to the specific methods or compositions described. It should also be understood that the terms used herein are for illustrative purposes only and are not intended to limit the embodiments.

[0051] Where a range of values ​​is provided, it should be understood that, unless explicitly indicated in the context, each intervening value up to one-tenth of a unit between the upper and lower limits of the stated range is also specifically disclosed. Each smaller range between any stated value or intervening value within the stated range and any other stated value or intervening value within that stated range is encompassed by the invention. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range in which either limit is included in the smaller range, neither limit is included in the smaller range, or both limits are included in the smaller range, subject to any specifically excluded limit within the stated range, is also encompassed by the invention. Where a stated range includes one or both limit values, the range excluding one or both of the limit values ​​that they include is also included by the invention.

[0052] Unless otherwise defined, technical and scientific terms used herein shall be interpreted as generally understood by those skilled in the art in which the invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, but several possible preferred methods and materials are described herein. All publications, patents, published patent applications, GenBank accession numbers and UniProt reference numbers referenced herein are incorporated herein by reference to disclose and describe the methods and / or materials with which the publications are cited.

[0053] When used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless explicitly indicated by the context. For example, a reference to “cell” includes multiple such cells, and a reference to “peptide” includes one or more peptides and their equivalents known to those skilled in the art, such as polypeptides.

[0054] The publications described herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention has no prior rights to such publications by prior art. Furthermore, the publication dates provided may differ from the actual publication dates which may need to be independently verified.

[0055] Unless otherwise specified, parts are measured by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius (°C), and pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations are used, including: bp = base pair; kb = kilobase; pl = picoliters; s or sec = seconds; min = minutes; h or hr = hours; AA or aa = amino acids; kb = kilobases; nt = nucleotides; pg = picograms; ng = nanograms; μg = micrograms; mg = milligrams; g = grams; kg = kilograms; dl or dL = deciliters; μl or μL = microliters; ml or mL = milliliters; l or L = liters; μM = micromoles; mM =millimole; M=moles; kDa=kilodaltons; im=intramuscular; ip=intraperitoneal; SC or SQ=subcutaneous; QD=daily; BID=twice daily; QW=once a week; QM=once a month; HPLC=high-performance liquid chromatography; BW=body weight; U=units; ns=not statistically significant; PBS=phosphate-buffered saline; PCR=polymerase chain reaction; HSA=human serum albumin; MSA=mouse serum albumin; DMEM=Dulbeco's Modification of Eagle's Medium; EDTA=ethylenediaminetetraacetic acid.

[0056] Throughout this disclosure, amino acids will be referenced by one-letter or three-letter codes.

[0057] Standard methods in molecular biology are described in the scientific literature (see, for example, Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), complex carbohydrate and protein expression (Vol. 3), and bioinformatics (Vol. 4)). Scientific literature describes protein purification methods including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, fusion protein production, and protein glycosylation (see, for example, Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).

[0058] Nomenclature for amino acid substitutions and deletions This disclosure provides variant polypeptides that include amino acid substitutions compared to the wild-type or parent polypeptide. The following nomenclature is used herein to refer to substitutions, deletions, or insertions. Residues may be designated herein by a one- or three-letter amino acid code of the native amino acid found in the wild-type molecule. In this disclosure, the numbering of amino acid residues in the human IL10 polypeptide reverse monomer is done by referring to the number of residues provided in SEQ ID NO:10. With respect to hIL10 mutein, substitutions are indicated herein by a one-letter amino acid code followed by the wt hIL10 (SEQ ID NO:10) amino acid position followed by the one-letter amino acid code being substituted. For example, hIL10 mutein having the modification "D25K" means that the aspartic acid (D) residue at position 25 of (SEQ ID NO:10) is substituted with a lysine (K) residue at this position. Deletions of amino acid residues are referred to as "des" or the symbol "Δ", followed by the amino acid residue and its position.

[0059] Immunoglobulin, Upper Hinge, and Fc Residue Numbering: Various numbering rules exist for the numbering of amino acid residues of immunoglobulins, including the Kabat numbering rules, Chothia numbering rules, EU numbering rules, and IMGT numbering rules. In the context of this disclosure, the numbering of amino acid residues of immunoglobulin molecules, including their domains, including the upper hinge and Fc domain (including the lower hinge, CH2 domain, and CH3 domain), is carried out in accordance with the EU numbering rules. The conversion of the EU numbering rules used herein to the Kabat numbering rules, Chothia numbering rules, or IMGT numbering rules will be readily understood by those skilled in the art. Dondelinger, et al. (2018) Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition Frontiers in Immunology Volume 9 Article#:2278.

[0060] definition Unless otherwise specified, the following terms are intended to have the meanings set forth below. Other terms are defined elsewhere throughout this specification.

[0061] about The term "about" refers to a value that is plus or minus 10% of any number given herein, for example, plus or minus 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of any number given herein. The term "about" also applies to any range of numbers given herein. Any value given herein is understood to be modified by the term "about," whether or not the term "about" is explicitly enumerated in relation to a given value.

[0062] ActivateWhere used herein, the term “activate” is used in reference to a receptor or receptor complex to directly reflect the biological effects resulting from the binding of an agonist ligand to a receptor in response to ligand binding and / or by its involvement in a multi-component signaling cascade. The terms “activate” or “be activated” are also used in reference to a cellular state in response to exposure of a cell to an activator.

[0063] Activation :As used herein, the term “activity” is used in reference to a molecule to describe its properties to a test system (e.g., an assay), or its biological or chemical properties (e.g., the degree of a molecule’s binding to another molecule), the effect of an active agent on a cell (e.g., cell activation), or the physical properties of a material or cell (e.g., modification of cell membrane potential). Examples of such biological functions include, but are not limited to, the catalytic activity of a biological active agent, its ability to stimulate intracellular signaling, gene expression, cell proliferation, and its ability to modulate immunological activities such as inflammatory responses. “Activity” is typically expressed as the level of biological activity per unit of the tested active agent, e.g., [catalytic activity] / [mg protein], [immunological activity] / [mg protein], international units (IU) of activity, [STAT3 phosphorylation] / [mg protein], [proliferation] / [mg protein], plaque-forming units (pfu), etc.As used herein, the term proliferative activity refers to the activity of an active agent that promotes cell proliferation and / or replication.

[0064] Administer / AdministrationThe terms “administer” and “administer” are used herein without distinction to refer to the act of contacting a subject, including, in vitro, in vivo, or ex vivo, with an active substance (e.g., an inverse monomer, a nucleic acid encoding an inverse monomer, a vector containing a nucleic acid encoding an inverse monomer (e.g., an expression vector), or engineered cells expressing an inverse monomer) or a pharmaceutical formulation containing one or more of the aforementioned active substances. The administration of the active substance may be achieved by any of the various methods recognized in the art, including, but not limited to, local administration, oral administration, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal delivery, transmucosal delivery, ionophoretic delivery, translymph node injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhalation (e.g., respiratory inhaler including dry powder inhaler), intraocular injection, intraabdominal injection, intrafocal injection, intraovarian injection, intracerebral or intracerebral injection, intraventricular infusion (ICVI), etc. The term “administration” includes contact of the active substance with cells, tissues or organs, and contact of the active substance with fluids in contact with cells, tissues or organs.

[0065] affinity As used herein, the term "affinity" refers to the degree of specific binding of a first molecule (e.g., a ligand) to a second molecule (e.g., a receptor), and the equilibrium dissociation constant (K). D ), the dissociation rate constant (K) between the molecule and its target off ) and the association rate constant (K) between the molecule and its target. on It is measured by the ratio of )

[0066] AgonistAs used herein, the term “agonist” refers to a first active substance that specifically binds to a second active substance (“target”) and interacts with the target to cause or promote increased activation of the target. In some cases, an agonist is an activator of a receptor protein that upregulates the expression of one or more genes, proteins, ligands, receptors, or biological pathways that can modulate, enhance, sensitize cells to activation by a second active substance, or, without limitation, result in the modulation of cellular activity, including cellular activation and / or proliferation or the cell cycle. In some embodiments, an agonist is an active substance that binds to a receptor, alters the state of the receptor, and produces a biological response that mimics the action of the receptor’s endogenous ligand. In some embodiments, an agonist is a modified form of a congener ligand that binds to its congener receptor and alters the state of the congener receptor in a biological response that mimics the biological effects of the interaction between a naturally occurring congener ligand and its congener receptor. The term “agonist” includes partial agonists, full agonists, and superagonists. An agonist may be described as a “full agonist” if such an agonist produces a substantially complete biological response (i.e., a response related to a naturally occurring ligand / receptor binding interaction). A “partial agonist” is a type of agonist that can produce a smaller maximal response to a target receptor than an endogenous agonist, and therefore has less than 100% of the maximal activity of the natural ligand to the target. In some embodiments, a superagonist, when evaluated at similar concentrations in an equivalent assay, exhibits a response less than 100% but greater than 10%, or greater than 20%, or greater than 30%, or greater than 40%, or greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90% of the evaluable quantitative or qualitative parameters of an endogenous agonist to a target receptor. A “superagonist” is a type of agonist that can produce a larger maximal response to a target receptor than an endogenous agonist, and therefore has more than 100% of the maximal activity of the natural ligand to the target.In some embodiments, superagonists, when evaluated at similar concentrations in equivalent assays, exhibit responses of over 110%, over 120%, over 130%, over 140%, over 150%, over 160%, or over 170% in evaluable quantitative or qualitative parameters of endogenous agonists to the target receptor. It should be noted that the biological effects associated with partial agonists, full agonists, or superagonists may differ not only in degree but also in type from the biological effects of endogenous agonists to the target receptor.

[0067] Antagonist As used herein, the terms “antagonist” or “inhibitor” refer to molecules that counteract the action of an agonist. Antagonists prevent, reduce, inhibit, or neutralize the activity of an agonist, and antagonists can also prevent, inhibit, or reduce the constitutive activity of a target, such as a target receptor, even in the absence of an identified agonist. Inhibitors are molecules that reduce, block, prevent, delay, inactivate, desensitize, or downregulate, for example, genes, proteins, ligands, receptors, biological pathways including immune checkpoint pathways, or cells.

[0068] Biological samples As used herein, the terms “biological specimen” or “sample” refer to a sample obtained from (or derived from) a subject. For example, a biological specimen includes material selected from the group consisting of body fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluids (e.g., vitreous fluid, aqueous humor), lymph, lymph node tissue, spleen tissue, bone marrow, and tumor tissue, which include immunoglobulin-enriched fractions or cell-type-specific enriched fractions derived from one or more such tissues.

[0069] equivalentAs used herein, the term “equivalent” is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, two measurements are considered “equivalent” if a first measurement of an evaluable quantitative parameter and a second measurement of the evaluable parameter do not deviate by a range that a person skilled in the art would recognize as not producing a statistically significant difference in effect between the two results in the given context. In some cases, measurements may be considered “equivalent” if one measurement deviates from another by less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 7%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%. In certain embodiments, a measurement is equivalent to a reference standard if it deviates from a reference standard by less than 15%, or less than 10%, or less than 5%.

[0070] Conservative amino acid substitutions As used herein, the term “conservative amino acid substitution” refers to an amino acid substitution that changes a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). Each of the following groups of amino acids is typically considered a conserved amino acid of one another: (1) hydrophobic amino acids: alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, and glycine; (2) polar amino acids: glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, and cysteine; (3) basic amino acids: lysine and arginine; and (4) acidic amino acids: aspartic acid and glutamic acid.

[0071] ~ corresponds toWhere used herein, the terms “correspondence” or “corresponding to” relating to amino acids or nucleotides within a polypeptide or polynucleotide, respectively, refer to the equivalent position (e.g., amino acid or nucleotide) of a reference polypeptide sequence or reference polynucleotide sequence when the reference sequence is aligned with a second sequence to maximize the percentage of sequence identity. For example, the “amino acid position corresponding to amino acid position [X]” of a particular IL10 polypeptide refers to the equivalent position based on alignment within other IL10 polypeptides containing IL10 derived from structural homologs and variants, or from different species (e.g., human IL10 and mouse IL10). The corresponding position is obtained based on the reference sequence, wild-type sequence, or parent sequence; for example, with respect to hIL10 mutein, the reference sequence may be the amino acid sequence of wild-type hIL10 (SEQ ID NO: 10).

[0072] ~ derived from As used herein, the term “derived from” in relation to a polypeptide, polynucleotide variant, or mutein means that the polypeptide, polynucleotide variant, or mutein has a sequence based on the sequence of a reference polypeptide (e.g., the amino acid sequence of wild-type hIL10 (SEQ ID NO: 10)) or a polynucleotide sequence (e.g., the cDNA encoding wt hIL10). The term “derived from” should not be understood as limiting the source or method by which the polypeptide, polynucleotide variant, or mutein is produced.

[0073] Effective concentration (EC)Where used herein, the term “effective concentration” or its abbreviation “EC” is used interchangeably to refer to the concentration of an active substance sufficient to produce a change in a given parameter of a test system. The abbreviation “E” refers to the magnitude of a given biological effect observed in a test system when the test system is exposed to the test active substance. The abbreviation “EC” is used when the magnitude of the response is expressed as a factor of the concentration of the test active substance ("C"). In the context of biological systems, the term Emax refers to the maximum magnitude of a given biological effect observed in response to the saturation concentration of the test active substance. The abbreviation EC can be followed by a subscript (e.g., EC 40 , EC 50 If provided, the subscript indicates the percentage of the Emax of the biological response observed at that concentration. For example, in a test system, the concentration of the test agent sufficient to induce the measurable biological parameter is 30% of the maximum level of such measurable biological parameter in response to such test agent. 30 It is called "EC". Similarly, the term "EC" 100 " is used to indicate the effective concentration of an active substance that yields the maximum (100%) response of a measurable parameter in response to such an active substance. Similarly, EC (commonly used in the field of pharmacodynamics) 50 The term "saturation concentration" refers to the concentration of an active substance sufficient to produce a change of half (approximately 50%) of the maximum value of a measurable parameter. The term "saturation concentration" refers to the maximum possible amount of a test active substance that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard temperature and pressure conditions. In pharmacodynamics, the saturation concentration of a drug is typically used to indicate a sufficient concentration of the drug such that all available receptors are occupied by the drug, and EC 50 This is the drug concentration that produces half the maximum effect.

[0074] concentratedAs used herein, the term “concentrated” means a sample that has not been naturally manipulated so that the species of interest (e.g., molecule or cell) is present at a higher concentration (e.g., at least 3 times, or at least 5 times, or at least 10 times, or at least 50 times, or at least 1000 times) than the concentration of the species in the starting sample, such as a biological sample (e.g., a sample in which the molecule is naturally present or present after administration), or (b) at a higher concentration than the environment in which the molecule was produced (e.g., recombinant bacterial or mammalian cells).

[0075] Extracellular domain As used herein, the term “extracellular domain” or its abbreviation “ECD” refers to the portion of a cell surface protein (e.g., a cell surface receptor) located outside the cell’s plasma membrane. A cell surface protein may be a transmembrane protein, a cell surface-associated protein, or a membrane-associated protein. A cell surface protein may be a multi-pass transmembrane protein having multiple non-adjacent extracellular domains.

[0076] Fusion proteinThe term "fusion protein" refers to a polypeptide comprising an amino acid sequence derived from a different protein or synthetic sequence that provides a different function. Examples of fusion proteins include, but are not limited to, a protein and a polypeptide comprising an immunogenic domain (e.g., diphtheria or tetanus toxin), a polypeptide domain for promoting expression (e.g., signal peptide), a sequence for promoting isolation and / or purification (e.g., chelate peptide), a polypeptide targeting domain (e.g., single-domain antibody), a protein with distinct additional (e.g., complementary, especially for complementary therapeutic) function, or a polypeptide domain for promoting a long duration of action in vivo (e.g., albumin). The domains of a fusion protein may be further linked by polypeptide linkers. The functional domains of a fusion protein may be provided in any N-terminus to C-terminus order. Fusion proteins are typically produced by translation of a single recombinant DNA sequence such that the protein and the additional functional domains of the fusion protein are covalently linked by peptide bonds.

[0077] identityAs used herein with respect to polypeptide sequences or DNA sequences, the term “identity” refers to subunit sequence identity between two molecules. Molecules are identical at a subunit position if the same monomeric subunit (i.e., the same amino acid residue or nucleotide) occupies the same subunit position within both molecules. The similarity between two amino acid sequences or two nucleotide sequences is a direct function of the number of identical positions. Generally, sequences are aligned to obtain highest-level matching. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the BLAST 2.0 algorithm described in Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul, et al. (1977) Nucleic Acids Res.25:3389-3402. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short strings of length W in the query sequence, which, when aligned with strings of the same length in the database sequence, match or satisfy a positive threshold score "T", where T is the neighboring string score threshold (Altschul, et al., hereafter). These initial neighboring string hits serve as seeds to initiate a search for longer HSPs containing them. String hits are then extended bidirectionally along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters "M" (reward score value for a matching pair of residues; always >0) and "N" (penalty score for mismatched residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix.(a) The extension of string hits in each direction is stopped if the cumulative alignment score decreases by amount X from its maximum achieved value, if the cumulative score becomes zero or less due to the accumulation of one or more negative score residue alignments, or (b) if the end of any sequence is reached. The BLAST algorithm parameters "W", "T", and "X" determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) works similarly but uses a string size of 28 ("W"), an expected value of 10 ("E"), M=1, N=-2, and comparison of both strands as defaults. For amino acid sequences, the BLASTP program uses a string size of 3 ("W"), an expected value of 10 ("E"), and the BLOSUM62 scoring matrix as defaults (see Henikoff & Henikoff, (1989) PNAS (USA) 89:10915-10919).

[0078] In an amount sufficient to produce a response Where used herein, the phrase “in an amount sufficient to produce a response” is used with respect to an amount of test activator sufficient to provide a detectable change in the level of an indicator measured before (e.g., baseline level) and after application of the test activator to a test system. In some embodiments, the test system is a cell, tissue, or organism. In some embodiments, the test system is an in vitro test system, such as a fluorescence assay. In some embodiments, the test system is an in vivo system involving the measurement of changes in parameter levels of cells, tissues, or organisms that reflect biological function before and after application of the test activator to the cells, tissues, or organisms. In some embodiments, the indicators reflect the biological function or developmental state of cells evaluated in the assay in response to the administration of a certain amount of test activator. In some embodiments, the test system involves the measurement of changes in indicator levels of cells, tissues, or organisms that reflect biological state before and after application of one or more test activators to the cells, tissues, or organisms. The term “in an amount sufficient to produce a response” may be sufficient to be a therapeutically effective dose, but may be more or less than a therapeutically effective dose.

[0079] Treatment is needed As used herein, the term “requiring treatment” refers to a determination made by a physician or other caregiver regarding a person who requires treatment or who may benefit from treatment. This determination is based on a variety of factors within the scope of the physician's or caregiver's expertise.

[0080] Preventive measures are necessary As used herein, the term “requiring prevention” means a determination made by a physician or other caregiver regarding a person who requires or may benefit from preventive care. This determination is based on a variety of factors within the scope of the physician's or caregiver's expertise.

[0081] inhibitors As used herein, the term “inhibitor” refers to a molecule that reduces, blocks, prevents, delays, inactivates, desensitizes, antagonistizes, or downregulates, for example, a gene, protein, ligand, receptor, or cell. An inhibitor may also be defined as a molecule that reduces, blocks, or inactivates the constitutive activity of a cell or organism.

[0082] intracellular domain As used herein, the term “intracellular domain” or its abbreviation “ICD” refers to a portion of a cell surface protein (e.g., a cell surface receptor) located inside the plasma membrane of a cell. An ICD may include the entire cytoplasmic portion of a transmembrane protein or membrane-associated protein, or an intracellular protein. A cell surface protein may be a multi-pass transmembrane protein having multiple non-proximal intracellular domains.

[0083] isolatedWhere used herein, the term “isolated” is used in reference to a molecule of interest that, if it exists in nature, is in an environment different from the environment in which it could naturally exist. “Isolated” means that the molecule of interest is substantially concentrated and / or is contained within a sample in which it is partially or substantially purified. Where the molecule does not exist in nature, “isolated” indicates that the molecule has been separated from the environment in which it was synthesized.

[0084] Ligand As used herein, the term “ligand” refers to a molecule that specifically binds to a receptor and causes a change in the receptor’s activity or a change in the receptor that results in a measurable response within a cell expressing that receptor. In one embodiment, the term “ligand” refers to a molecule or complex that can act as an agonist or antagonist of a receptor. As used herein, the term “ligand” encompasses both native and synthetic ligands. “Ligand” also encompasses cytokines and small molecules of antibodies, peptide mimes. The complex of a ligand and a receptor is called a “ligand-receptor complex.” A ligand may consist of one domain of a polyprotein or fusion protein (e.g., one domain of an antibody / ligand fusion protein).

[0085] Linker As used herein, the term “linker” refers to a molecule used to link a first and a second heterologous molecule. In some embodiments, the linker may be a chemical linker. In some embodiments, the linker is a “polypeptide linker” and represents a polypeptide used to link a functional subunit of a polypeptide (e.g., a fusion protein) which consists of multiple functional domains or functional subunits.

[0086] ModifiedAs used herein, the term “modified” refers to a molecule, such as a polypeptide, whose structure has been altered compared to its unmodified parent molecule. Modified polypeptides typically retain one or more activities or functions of their unmodified parent molecule. For example, the reverse monomers of this disclosure may activate IL10 signaling in cells expressing the IL10 receptor as part of a homodimer, but may have improved properties compared to the unmodified polypeptide. The term “modified” includes amino acid substitutions not present in the reverse structure of the parent or wild-type IL10 polypeptide or wild-type monomer polypeptide, and includes variants and mutains of IL10 reverse monomer polypeptides.

[0087] Adjust As used herein, terms such as “modulate” and “modulation” refer to the ability of a test substance to elicit a positive or negative, direct or indirect, response in a system, including a biological system or biochemical pathway. The term “modulator” includes both agonists (including partial agonists, complete agonists, and superagonists), inhibitors, and antagonists.

[0088] nucleic acid As used herein, the terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably to refer to polymeric forms of nucleotides of any length (including deoxyribonucleotides or ribonucleotides). Non-exclusive examples of polynucleotides include linear and cyclic nucleic acids, messenger RNA (mRNA), small nuclear RNA (snRNA), small interfering RNA (siRNA), guide RNA (gRNA) complementary DNA (cDNA), recombinant polynucleotides, recombinant viral vectors or recombinant nonviral vectors, recombinant viral expression vectors or recombinant nonviral expression vectors, hybridization probes, PCR primers, and the like.

[0089] One or more amino acid substitutionsAs used herein, the term “one or more amino acid substitutions” refers to a single amino acid substitution or one, two, three, four, five or more amino acid substitutions compared to a reference sequence. In some embodiments, the reference sequence is the wild-type hIL10 monomer or the inverse hIL10 of this disclosure.

[0090] Functionally linkedThe term “functionally linked” is used herein to refer to a relationship between a first or second component molecule, typically a polypeptide or nucleic acid, that is positioned within a construct such that at least one of the functions of the component molecules is retained in the construct. However, functional linking can result in a construct exhibiting either positively or negatively regulated activity of the individual components of the construct. For example, functional linking of a polyethylene glycol (PEG) molecule to a wild-type protein may result in a construct in which the biological activity of the protein is reduced compared to the wild-type molecule, but nevertheless, these two are considered functionally linked. When the term “functionally linked” is applied to a relationship between multiple nucleic acid sequences encoding different functions, the multiple nucleic acid sequences provide a nucleic acid that, when combined into a single nucleic acid molecule, for example, when introduced into a cell using recombination techniques, can result in the transcription and / or translation of one or more of the nucleic acid sequences, as well as the expression of one or more proteins in the cell. For example, a nucleic acid sequence encoding a signal sequence can be considered functionally linked to the polypeptide-coding DNA if it results in the expression of a preprotein, thereby promoting the secretion of the polypeptide; a promoter or enhancer can be considered functionally linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site can be considered functionally linked to a coding sequence if it is positioned to facilitate translation. Generally, in the context of nucleic acid molecules, the term “functionally linked” means that the linked nucleic acid sequences are contiguous and, in the case of a molecule’s secretory leader or associated subdomain, contiguous and in the read-through phase. However, certain genetic elements, such as enhancers, can function at a certain distance and do not need to be contiguous with respect to the sequence that provides their effect, but can nevertheless be considered functionally linked.

[0091] Parent polypeptideWhere used herein, the terms “parent polypeptide” or “parent protein” are used interchangeably to indicate the source of a second polypeptide (e.g., a derivative, mutain, or variant) that is modified with respect to a first “parent” polypeptide. In some cases, the parent polypeptide is a wild-type protein, or a naturally occurring form of protein. In some cases, the parent polypeptide may be a naturally occurring protein in a modified form that is further modified. The term parent polypeptide may also be used interchangeably with “reference polypeptide.”

[0092] Partial agonist As used herein, the term “partial agonist” refers to a molecule that specifically binds to a given receptor and activates it, but only partially activates the receptor compared to a full agonist. Partial agonists may exhibit both agonist and antagonistic effects. For example, in the presence of both a full agonist and a partial agonist, a partial agonist may act as a competitive inhibitor of the full agonist by competing with the full agonist for receptor binding, resulting in a net reduction in receptor activation compared to contact between the receptor and the full agonist in the absence of the partial agonist. Partial agonists can be used to activate a receptor to give a subject a desired sub-maximum response in the absence of endogenous ligand or in the presence of depleted levels of endogenous ligand. Partial agonists can be used to reduce receptor overstimulation in the presence of an excess amount of endogenous ligand. The maximum response (E) induced by a partial agonist max This is called the intrinsic activity and can be expressed on a percentage scale from which a complete agonist yields a 100% response. Partial agonists, when evaluated at similar concentrations in a given assay system, may have activity levels of more than 10% but less than 100%, or more than 20% but less than 100%, or more than 30% but less than 100%, or more than 40% but less than 100%, or more than 50% but less than 100%, or more than 60% but less than 100%, or more than 70% but less than 100%, or more than 80% but less than 100%, or more than 90% but less than 100%.

[0093] Percent Identity As used herein, the term “percent (%) sequence identity” as used in relation to nucleic acids or polypeptides refers to a sequence having at least 50% sequence identity with a reference sequence. Alternatively, percent sequence identity can be any integer between 50% and 100%. In some embodiments, a sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to a reference sequence, as determined by BLAST using standard parameters as described below.

[0094] In sequence comparison, typically one sequence acts as a reference sequence, and the test sequence is compared against the reference sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.

[0095] The comparison window includes a reference to any one of several consecutive segments, for example, a segment of at least 10 residues. In some embodiments, the comparison window has 10 to 600 residues, for example, about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, and the sequences can be compared to reference sequences of the same number of consecutive positions after the two sequences have been optimally aligned.

[0096] Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1977) Nucleic Acids Res.25:3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short strings of length W within the query sequence, which, when aligned with strings of the same length in the database sequence, match or satisfy some positive threshold score T, where T refers to the neighbor string score threshold (Altschul, et al., ibid.). These initial neighbor string hits serve as seeds to initiate a search for longer HSPs containing them. Next, string hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score value for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of string hits in each direction stops if the cumulative alignment score decreases by X from its maximum achieved value, if the cumulative score becomes zero or less due to the accumulation of one or more negative score residue alignments, or if the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses a string size of 28 (W), an expected value of 10 (E), M=1, N=-2, and comparison of both strands as defaults.For amino acid sequences, the BLASTP program uses a string size of 3 (W), an expected value of 10 (E), and a BLOSUM62 scoring matrix as defaults (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0097] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indicator of the probability that the match between two nucleotide or amino acid sequences occurs by chance. For example, a smallest sum probability of less than about 0.01, more preferably about 10, in the comparison between a test amino acid sequence and a reference amino acid sequence is desirable. -5 Less than, most preferably about 10 -20 If the value is less than [a certain value], the amino acid sequence is considered to be similar to the reference sequence.

[0098] polypeptide Where used herein, the terms “polypeptide,” “peptide,” and “protein” refer to polymeric forms of amino acids of any length. Polypeptides may include genetically encoded and unencoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified polypeptide backbone. The term polypeptide includes fusion proteins. The term polypeptide also includes fusion proteins of a first polypeptide and a second heterologous polypeptide carrier protein (e.g., human serum albumin (HSA)).

[0099] PreventAs used herein, the terms “prevent,” “preventing,” and “prevention” refer to a series of actions initiated on a subject before the onset of a disease, disorder, condition, or its symptoms, in order to temporarily or permanently prevent, suppress, inhibit, or reduce the risk of the subject developing a disease, disorder, condition, etc. (for example, as determined by the absence of clinical symptoms), or to delay its onset. A series of actions to prevent a disease, disorder, or condition in a subject is typically applied in relation to a subject who is susceptible to developing a disease, disorder, or condition due to genetic, empirical, or environmental factors that cause the development of a particular disease, disorder, or condition.

[0100] receptorAs used herein, the term “receptor” refers to a polypeptide having a domain that specifically binds to a ligand, and ligand binding results in a change to at least one biological property of the polypeptide. In some embodiments, the receptor is a cell membrane-associated protein comprising an extracellular domain (ECD) and a membrane-associated domain that helps fix the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane-spanning polypeptide comprising an intracellular domain (ICD) and an extracellular domain (ECD) linked by a transmembrane-spanning domain typically called a transmembrane domain (TM). Binding of a homologous ligand to a receptor results in a change to the receptor, resulting in a measurable biological effect. In some cases, if the receptor is a transmembrane-spanning polypeptide comprising the ECD, TM, and ICD, ligand binding to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to ligand binding to the ECD. In some embodiments, receptors are components of a multicomponent complex, which, when assembled, leads to intracellular signaling. For example, a ligand may bind to a cell surface receptor not solely associated with intracellular signaling, but upon ligand binding, promotes the formation of a heteromultimer (including heterodimers, heterotrimers, etc.) or homomultimer (including homodimers, homotrimers, homotetramers, etc.) complex that produces measurable biological effects within the cell, such as activation of an intracellular signaling cascade (e.g., the Jak / STAT pathway). In some embodiments, the receptor is a transmembrane-spanning single-chain polypeptide comprising an ECD domain, a TM domain, and an ICD domain, the ECD domain, TM domain, and ICD domain derived from the same or different naturally occurring receptor variants or their synthetic functional equivalents.

[0101] RecombinantWhere used herein, the term “recombinant” is used as an adjective referring to a method of modifying polypeptides, nucleic acids, or cells using recombinant DNA technology. “Recombinant protein” is a protein produced using recombinant DNA technology and is often abbreviated with a lowercase “r” preceding the protein name to indicate how the protein was produced (for example, recombinant human growth hormone is commonly abbreviated as “rhGH”). Similarly, cells are called “recombinant cells” when they are modified by the incorporation of exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids, etc.) using recombinant DNA technology (e.g., transfection, transduction, infection). Techniques and protocols for recombinant DNA technology are well known in the art and can be found, for example, in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0102] responseFor example, the term “response” of a cell, tissue, organ, or organism encompasses quantitative or qualitative changes in evaluable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression levels, gene expression rates, energy consumption rates, levels or states of differentiation) that correlate with exogenous agents or internal mechanisms, such as activation, stimulation, or treatment by genetic programming, or contact with exogenous agents or internal mechanisms, such as genetic programming. In certain contexts, terms such as “activation” and “stimulation” refer to cellular activation regulated by internal mechanisms and by external or environmental factors, while terms such as “inhibition” and “downregulation” refer to the opposite effect. “Response” can be evaluated in vitro by assay systems, flow cytometry assays, surface plasmon resonance, enzyme activity, mass spectrometry, amino acid or protein sequencing techniques, etc. The "response" can be assessed in vivo quantitatively by evaluating objective physiological parameters such as body temperature, body weight, tumor volume, blood pressure, and the results of X-ray or other imaging techniques, or qualitatively by reporting subjective changes in well-being, depression, agitation, or pain. In some embodiments, the level of T cell activation in response to administration of the test agent can be determined by flow cytometry, as described, where it is determined by the level of one or more STATs (e.g., STAT1, STAT3, or STAT5) according to methods well known in the art.

[0103] Significantly reduced bindingAs used herein, the term “showing markedly reduced binding” is used in reference to a variant of a first molecule (e.g., ligand or antibody) that shows a markedly reduced affinity for a second molecule (e.g., receptor or antigen) compared to the parent form of the first molecule. With respect to an antibody variant, an antibody variant “shows markedly reduced binding” if the affinity of the variant antibody to the antigen, or the affinity of the variant to the native form of the receptor, is less than 20%, or about 10%, or about 8%, or about 6%, or about 4%, or about 2%, or about 1%, or about 0.5% of that of the parent antibody from which the variant originates. With respect to a variant ligand or ligand mutein, if the affinity of the variant ligand or mutein to the receptor is less than 20%, or about 10%, or about 8%, or about 6%, or about 4%, or about 2%, or about 1%, or about 0.5%, of the parent ligand from which the variant ligand is derived, the variant ligand or mutein "shows markedly reduced binding." Similarly, with respect to a variant receptor, if the affinity of the variant receptor to the ligand is less than 20%, or about 10%, or about 8%, or about 6%, or about 4%, or about 2%, or about 1%, or about 0.5%, of the parent receptor from which the variant receptor is derived, the variant receptor "shows markedly reduced binding."

[0104] Specific bindingAs used herein, the term “specifically binds” refers to the degree of affinity that a first molecule exhibits for a second molecule. In the context of binding pairs (e.g., ligand / receptor, antibody / antigen), the first molecule of a binding pair is said to bind specifically to the second molecule of a binding pair if the first molecule does not bind in significant amounts to other components present in the sample. The first molecule of a binding pair is said to bind specifically to the second molecule of a binding pair if its affinity for the second molecule is at least twice, or at least five times, or at least ten times, or at least twenty times, or at least one hundred times, its affinity for other components present in the sample. Specific binding can be evaluated using techniques known in the art, without limitation, including competitive ELISA assays, radioactive ligand binding assays (e.g., saturated binding, Scatchard plots, nonlinear curve fitting programs, and competitive binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET); liquid-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR) and immunoprecipitation); and solid-phase ligand binding assays (e.g., multi-well plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays)) and surface plasmon resonance assays (e.g., using commercially available instruments, e.g., Biacore 8K, Biacore 8K+, Biacore S200, Biacore T200 (Cytiva, 100 Results Way, Marlborough MA 01752)). See Drescher et al., (2009) Methods Mol Biol 493:323-343.

[0105] subjectThe terms “recipient,” “individual,” “subject,” and “patient” are used without distinction herein and refer to any mammalian subject for which diagnosis, treatment, or therapy is desired. “Mammal” for therapeutic purposes refers to any animal classified as a mammal, including humans, domesticated and livestock, as well as animals in zoos, sports, or as pets, such as dogs, horses, cats, cows, sheep, goats, and pigs. In some aspects, a mammal is a human.

[0106] Essentially pure As used herein, the term “substantially pure” means that the components of the composition constitute about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% of the total content of the composition. “Substantially pure” protein constitutes about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% of the total content of the composition.

[0107] ~ is suffering from As used herein, the term “suffering from” means, without limitation, a decision made by a physician regarding a subject whether the subject requires treatment or would benefit from treatment, based on available objective or subjective information accepted in the field of identifying the presence of a disease, disorder or condition in a subject, including, but not limited to, X-ray, CT scan, conventional laboratory diagnostic tests (e.g., blood counts), genomic data, protein expression data, and immunohistochemical tests.

[0108] T cells As used herein, the term “T-cell” or “T cell” is used in its conventional sense to refer to lymphocytes that differentiate in the thymus. In some aspects, the term T cell is used, without limitation, to refer to naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, for example, T H 1. T H 2, TH 9, T H 11, T H 22, T FH ;regulatory T cells, e.g. R 1. Tregs, inducible Tregs; memory T cells, e.g., central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TILs), and, without limitation, engineered variants of such T cells, including CAR-T cells, recombinant modified TILs, and TCR-manipulated cells.

[0109] Terminus / Terminal When used herein in relation to the structure of a polypeptide, “N-terminus” (or “amino-terminus”) and “C-terminus” (or “carboxyl-terminus”) refer to the extreme amino-terminus and carboxyl-terminus of the polypeptide, respectively, and the terms “N-terminus” and “C-terminus” refer to the relative positions of the amino acid sequence of the polypeptide to the N-terminus and C-terminus, respectively, and the N-terminus and C-terminus may contain residues, respectively. “Immediately N-terminus” refers to the position of a first amino acid residue relative to a second amino acid residue in a consecutive polypeptide sequence, where the first amino acid is closer to the N-terminus of the polypeptide. “Immediately C-terminus” refers to the position of a first amino acid residue relative to a second amino acid residue in a consecutive polypeptide sequence, where the first amino acid is closer to the C-terminus of the polypeptide.

[0110] Therapeutic effective doseAs used herein, the term “therapeutic dose” refers to the amount of an active substance that, when administered to a subject, either alone or as part of a pharmaceutical composition or therapeutic regimen, in a single dose or as part of a series of doses, produces a positive effect on any quantitative or qualitative symptom, aspect or feature of a disease, disorder, or condition. The therapeutic dose may be determined by measuring the relevant physiological effects and may be adjusted in relation to the administration regimen and in accordance with a diagnostic analysis of the subject’s condition. Parameters for evaluation to determine the therapeutic dose of an active substance are determined by a physician using diagnostic criteria acceptable in the art, including, but not limited to, age, weight, sex, overall health status, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, and X-rays. Alternatively, to determine whether a therapeutically effective dose of the active ingredient has been administered to the subject, other parameters commonly evaluated in the clinical setting, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptoms, aspects, or characteristics of the disease, disorder, or condition, reduction of biomarkers (e.g., inflammatory cytokines, IFN-γ, granzymes, etc.), serum tumor markers, improvement, extension of survival, extension of progression-free survival, extension of time to progression, extension of time to treatment failure, extension of event-free survival, extension of time to next treatment, improvement of objective response rate, improvement of response duration, reduction of tumor burden, complete response, partial response, stable disease, etc., may be monitored. In one embodiment, a therapeutically effective dose is the amount of the active ingredient that, when used alone or in combination with another active ingredient, results in improvement to any quantitative or qualitative symptom, aspect, or characteristic of the disease, disorder, or condition, and does not result in any irreversible serious adverse event during the administration of the active ingredient to a mammalian subject.

[0111] TreatThe terms “treat,” “treating,” and “treatment” refer to a series of actions initiated with respect to a subject in response to a diagnosis that the subject is suffering from a disease, disorder, or condition or its symptoms (such as bringing the subject into contact with a pharmaceutical composition containing an inverse monomer and / or its dimer alone or in combination with an adjuvant), the series of actions initiated to temporarily or permanently eliminate, reduce, suppress, alleviate or improve (a) the underlying cause of such disease, disorder, or condition affecting the subject; and / or (b) at least one of at least one of the symptoms associated with such disease, disorder, or condition. In some embodiments, treating includes a series of actions performed with respect to a subject suffering from a disease, the series of actions resulting in inhibition of the disease in the subject (e.g., stopping the onset of the disease, disorder, or condition, or improving one or more of the symptoms associated therewith). In some embodiments, the term “treat” is used to refer to a process of actions that slow the progression of a disease, disorder, or condition from an existing condition to a more harmful condition.

[0112] variantThe terms “variant,” “protein variant,” “variant protein,” or “variant polypeptide” are used herein without distinction to refer to a polypeptide that differs from a parent polypeptide by modification, substitution, or deletion of at least one amino acid. The parent polypeptide may be a naturally occurring polypeptide, a wild-type (WT) polypeptide, or a modified (e.g., reverse) WT polypeptide. The term “variant polypeptide” may refer to the polypeptide itself, a composition containing the polypeptide, or a nucleic acid sequence encoding it. In some embodiments, a variant polypeptide may have about 1 to about 10, or about 1 to about 8, or about 1 to about 7, or about 1 to about 5, or about 1 to about 4, or about 1 to about 3, or 1 to 2 amino acid modifications, substitutions, or deletions, or single amino acid modifications, substitutions, or deletions, compared to the parent polypeptide. A variant may be at least about 99%, or at least about 98%, or at least about 97%, or at least about 95%, or at least about 90% identical to the parent polypeptide from which the variant is derived. The term "variant" also includes nucleic acid molecules that encode proteins or polypeptides that have altered or modified amino acid sequences compared to the parent polypeptide.

[0113] Wild type In this specification, "wild-type," "WT," or "natural" means the naturally occurring amino acid or nucleotide sequence, including allelic mutations. Wild-type proteins, polypeptides, antibodies, immunoglobulins, IgG, etc., have an amino acid or nucleotide sequence that has not been modified by humans.

[0114] For clarity and brevity, the individual embodiments described separately herein may be combined in any way without limitation. Accordingly, this disclosure includes one or more combinations, or all combinations, of the embodiments described herein, as if every possible combination were disclosed individually and expressly. This also applies to any partial combination of the embodiments disclosed herein, and as a result, this disclosure includes one or more partial combinations, or all partial combinations, of the embodiments described herein, as if every possible partial combination were disclosed individually and expressly.

[0115] inverse monomer This disclosure provides inverse monomers of IL10, including inverse monomers of hIL10. In some embodiments, the inverse monomers of IL10 are derived from the sequence of a mature IL10 (human or mouse) monomer. The canonical 160-amino acid sequence (corresponding to amino acids 19-178 of the preprotein) of a mature ("wild-type") human IL10 monomer without a signal sequence (UniProt reference number P22301) is as follows: It has TIFF2026519565000014.tif23128.

[0116] The canonical amino acid sequence of a mature mouse IL10 protein monomer lacking a signal sequence (UniProt reference number P18893) (corresponding to amino acids 19-178 of the preprotein) is as follows: The filename is TIFF2026519565000015.tif19128.

[0117] Based on its crystal structure, wild-type IL10 contains six alpha-helical domains. See PDB DOI:10.2210 / pdb2ILK / pdb. The amino acid sequences of the helices and their positions, compared with SEQ ID NO:11, are shown in Table 1 below.

[0118] (Table 1) Alpha helix of hIL10 TIFF2026519565000016.tif52152

[0119] The terms “IL10 reverse monomer” and “IL10 reverse monomer” are used interchangeably to refer to IL10 variants containing subsequences derived from wild-type IL10 that have been rearranged compared to the natural N-terminus-C-terminus order of such subsequences present in the naturally occurring IL10 monomer. For example, in some embodiments, one or more helices of IL10 are rearranged to their relative positions to a contiguous sequence of such helices in the wild-type IL10 sequence. In some embodiments of the reverse monomer of this disclosure, a polypeptide sequence containing helices 5 and 6 is provided on the N-terminal side of a polypeptide sequence containing helices 1-4. In some embodiments, the reverse IL10 monomer contains a polypeptide containing helices 5 and 6 (i.e., helices 5, 6, 1, 2, 3, and 4 in amino-to-carboxyl order) located on the N-terminal side of a polypeptide containing helices 1, 2, 3, and 4 in amino-to-carboxyl order. In some embodiments, the individual helices are linked by one or more independent amino acid linkers or polypeptide linkers. In some embodiments, the linker may contain a polypeptide derived from a wild-type IL10 sequence. In some embodiments, the linker may contain a heterologous or synthetic amino acid sequence or polypeptide sequence. In some embodiments, the inverse monomer may further contain amino acid sequences at the N-terminal side of the first helix (e.g., helix 5) and the C-terminal side of the last helix (e.g., helix 4). The additional N-terminal and C-terminal amino acid sequences may include a native or wild-type IL10 sequence that is typically located at the N-terminal or C-terminal side of a particular helix.

[0120] In some embodiments, the inverse monomer polypeptide comprises the amino acid sequence of formula 1: [A]-L1 x -[B]-L2-[C]-L3-[D]-L4 y -[E]-L5-[F](1) During the ceremony, x and y are selected independently of 0 (non-existent) or 1 (existent), and L1 to L5 refer to linkers 1 to 5, respectively.

[0121] In some embodiments, [A] corresponds to helix 5 of wild-type IL10, which corresponds to amino acid residues 118-131 of hIL10 (numbered according to SEQ ID NO: 11). In some embodiments, [A] is the amino acid sequence Includes TIFF2026519565000017.tif4128. In some embodiments, [A] comprises a variant of SEQ ID NO:1 having one or two amino acid substitutions compared to SEQ ID NO:1. In some embodiments, the variant sequence substantially retains the helical structure of [A] compared to the wild-type helix.

[0122] In some embodiments, x=0 (meaning no linker, L1 is not present) or x=1 (L1 is present), and L1 comprises a polypeptide linker of any preferred length, e.g., about 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 amino acids. In some embodiments, L1 comprises 1, 2, 3, 4, or 5 amino acids. In some embodiments, L1 comprises a GS linker as described herein. In some embodiments, L1 is the amino acid glutamine (Q).

[0123] In some embodiments, [B] corresponds to helix 6 of wild-type IL10, which corresponds to amino acid residues 133-159 of hIL10 (numbered according to SEQ ID NO: 11). In some embodiments, [B] is the amino acid sequence Includes TIFF2026519565000018.tif4128. In some embodiments, [B] includes a variant of SEQ ID NO:2 having one or two amino acid substitutions compared to SEQ ID NO:2. In some embodiments, the variant sequence substantially retains the helical structure of [B] compared to the wild-type helix.

[0124] In some embodiments, L2 comprises a linker of any preferred length, for example, about 1-40, 1-30, 1-25, 1-20, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-25, 5-20, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-25, or 10-20 amino acids. In some embodiments, L2 comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, L2 comprises a GS linker as described herein. In some embodiments, L2 comprises an amino acid sequence This polypeptide contains TIFF2026519565000019.tif4128. In some embodiments, L2 is an amino acid sequence This is a polypeptide containing TIFF2026519565000020.tif4128.

[0125] In some embodiments, [C] corresponds to helix 1 of wild-type IL10, corresponding to amino acid residues 18-41 of hIL10 (numbered according to SEQ ID NO: 11). In some embodiments, [C] corresponds to the amino acid sequence Includes TIFF2026519565000021.tif4128. In some embodiments, [C] includes a variant of SEQ ID NO:3 having one or two amino acid substitutions compared to SEQ ID NO:3. In some embodiments, the variant sequence substantially retains the helical structure of [C] compared to the wild-type helix.

[0126] In some embodiments, L3 comprises a linker of any preferred length, for example, about 1-40, 1-30, 1-25, 1-20, 1-10, 2-50, 2-40, 2-30, 2-20, 2-10, 3-50, 3-40, 3-30, 3-20, 3-10, 4-50, 4-40, 4-30, 4-20, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, or 5-10 amino acids. In some embodiments, L3 comprises 4, 5, 6, 7, 8, 9, 10, or 11 amino acids. In some embodiments, L3 comprises a GS linker as described herein. In some embodiments, L3 is a polypeptide comprising the amino acid sequence QLDNLLL (SEQ ID NO: 8).

[0127] In some embodiments, [D] corresponds to helix 2 of wild-type IL10, corresponding to amino acid residues 49-58 of hIL10 (numbered according to SEQ ID NO: 11). In some embodiments, [D] corresponds to the amino acid sequence Includes TIFF2026519565000022.tif4128. In some embodiments, [D] includes a variant of SEQ ID NO:4 having one or two amino acid substitutions compared to SEQ ID NO:4. In some embodiments, the variant sequence substantially retains the helical structure of [D] compared to the wild-type helix.

[0128] In some embodiments, y=0 (L4 is absent) or y=1, where L4 comprises a polypeptide linker. The L4 linker may be of any preferred length, e.g., about 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 amino acids. In some embodiments, L4 comprises 1, 2, 3, 4, or 5 amino acids. In some embodiments, L4 comprises a GS linker as described herein. In some embodiments, L4 is the amino acid glycine (G). In some embodiments, L4 comprises the amino acid sequence "GY".

[0129] In some embodiments, [E] corresponds to helix 3 of wild-type IL10, corresponding to amino acid residues 60-82 of hIL10 (numbered according to SEQ ID NO: 11). In some embodiments, [E] is the amino acid sequence Includes TIFF2026519565000023.tif4128. In some embodiments, [E] includes a variant of SEQ ID NO:5 having one or two amino acid substitutions compared to SEQ ID NO:5. In some embodiments, the variant sequence substantially retains the helical structure of [E] compared to the wild-type helix.

[0130] In some embodiments, L5 includes a linker. The linker L5 may be of any preferred length, e.g., about 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, or about 1-5 amino acids. In some embodiments, L5 includes 1, 2, 3, 4, 5, 6, or 7 amino acids. In some embodiments, L5 includes a GS linker as described herein. In some embodiments, L5 is a polypeptide comprising the amino acid sequence QDPD (SEQ ID NO: 9).

[0131] In some embodiments, [F] corresponds to helix 3 of wild-type IL10, corresponding to amino acid residues 87-108 of hIL10 (numbered according to SEQ ID NO: 11). In some embodiments, [F] corresponds to the amino acid sequence Includes TIFF2026519565000024.tif4128. In some embodiments, [F] includes a variant of SEQ ID NO:6 having one or two amino acid substitutions compared to SEQ ID NO:6. In some embodiments, the variant sequence substantially retains the helical structure of [F] compared to the wild-type helix.

[0132] In some embodiments, the domains of Equation 1 are shown in Table 2.

[0133] (Table 2) Reference sequences of domains [A], [B], [C], [D], [E], and [F] of formula 1: TIFF2026519565000025.tif51148

[0134] Each of the linkers described above may be independently selected as the wild type, i.e., as the amino acid sequence of the wild-type hIL10 sequence that links the two helices. Alternatively, one or more linkers may be variants of the wild-type linker sequence, i.e., variants having one, two or more amino acid substitutions, deletions, or insertions compared to the wild-type linker sequence. In some embodiments, L1 is the amino acid glutamine (Q). In some embodiments, L2 is the amino acid sequence This polypeptide contains TIFF2026519565000026.tif4128. In some embodiments, L2 is an amino acid sequence The polypeptide contains TIFF2026519565000027.tif4128. In some embodiments, L3 is a polypeptide containing the amino acid sequence QLDNLLL (SEQ ID NO: 8). In some embodiments, L4 is the amino acid glycine ("G"). In some embodiments, L4 contains the amino acid sequence "GY". In some embodiments, L5 is a polypeptide containing the amino acid sequence QDPD (SEQ ID NO: 9).

[0135] In some embodiments, one or more of L1, L2, L3, L4, and / or L5 (e.g., all of them) include a linker with a sequence different from the wild-type linker sequence. For example, in some embodiments, each of L1, L2, L3, L4, and / or L5 includes a GS-linker. The linker can be readily selected and may be of any preferred length, e.g., 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids. Examples of linkers useful in the implementation of this disclosure include, but are not limited to, glycine polymers (G)n where is an integer from 1 to 50. Examples of linkers useful in the implementation of this disclosure include, but are not limited to, glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers also referred to herein as "GS-linkers." Because glycine polymers and glycine-serine polymers are relatively unstructured, they can function as flexible tethers between the domains or subunits of polypeptides. In some embodiments, the GS-linker is of the formula: TIFF2026519565000028.tif31151 and selected polymers of combinations thereof, where m, n, and o are independently selected integers from 1 to 20, e.g., 1 to 18, 2 to 16, 3 to 14, 4 to 12, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of GS-linkers include, without limitation, Examples include TIFF2026519565000029.tif44149 and its polymers.

[0136] In some embodiments, the inverse monomer polypeptide contains or comprises an amino acid sequence derived from human IL10. In some embodiments, the inverse monomer polypeptide contains or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:10. TIFF2026519565000030.tif18128

[0137] In some embodiments, the inverse monomer polypeptide comprises or consists of an amino acid sequence having sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with respect to SEQ ID NO:10.

[0138] In some embodiments, the inverse monomer polypeptide contains or comprises an amino acid sequence derived from human IL10. In some embodiments, the inverse monomer polypeptide contains or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the following SEQ ID NO:10 or SEQ ID NO:11. TIFF2026519565000031.tif18128

[0139] In some embodiments, the inverse monomer polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the following SEQ ID NO:10 or SEQ ID NO:11.

[0140] In some embodiments, the present disclosure provides polypeptides comprising the following, in order from amino to carboxyl: (a) Below: (i) Amino acid residues 117-158 of human IL-10, numbered according to mature human IL-10 (SEQ ID NO: 20) TIFF2026519565000032.tif4135; (ii) Amino acid residues 117-159 of human IL-10 TIFF2026519565000033.tif4137; and (iii) Amino acid residues 117-160 of human IL-10 A first polypeptide comprising an amino acid sequence selected from the group consisting of TIFF2026519565000034.tif11128, (b) Linkers where x = 0 (no linker exists) or 1 (a linker exists) x , Furthermore (c) Below: (i) Amino acid residues 1-116 of human IL-10, numbered according to mature human IL-10 (SEQ ID NO: 20) TIFF2026519565000035.tif17139; (ii) Amino acid residues 2-116 of human IL-10 TIFF2026519565000036.tif17140; (iii) Amino acid residues 3-116 of human IL-10 TIFF2026519565000037.tif17139; (iv) Amino acid residues 4-116 of human IL-10 TIFF2026519565000038.tif17140; (v) Amino acid residues 5-116 of human IL-10 TIFF2026519565000039.tif17140. (vi) Amino acid residues 6-116 of human IL-10 TIFF2026519565000040.tif17140; (vii) Amino acid residues 7-116 of human IL-10 TIFF2026519565000041.tif17140; (viii) Amino acid residues 8-116 of human IL-10 TIFF2026519565000042.tif18140; (ix) Amino acid residues 9-116 of human IL-10 TIFF2026519565000043.tif17140; (x) Amino acid residues 10-116 of human IL-10 TIFF2026519565000044.tif17138; (xi) Amino acid residues 11-116 of human IL-10 TIFF2026519565000045.tif17138; and (xii) Amino acid residues 12-116 of human IL-10 A second polypeptide comprising an amino acid sequence selected from the group consisting of TIFF2026519565000046.tif17138.

[0141] In some embodiments, the fusion polypeptide comprises the following, in the order of amino to carboxyl: (i) A first polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to amino acid residues 117-158 of human IL-10 (SEQ ID NO:12), and (ii) A second polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:19.

[0142] In some embodiments, the fusion polypeptide comprises the following, in the order of amino to carboxyl: (i) A first polypeptide comprising amino acid residues 117-158 of human IL-10 (SEQ ID NO:12), or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% of an amino acid sequence having sequence identity SEQ ID NO:12, and (ii) A second polypeptide comprising an amino acid sequence having sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with respect to SEQ ID NO:19.

[0143] In some embodiments, the first polypeptide contains one, two, or three amino acid substitutions compared to SEQ ID NO:12. In some embodiments, the second polypeptide contains one, two, or three amino acid substitutions compared to SEQ ID NO:19.

[0144] In some embodiments, the first and second polypeptides are covalently linked by a peptide bond (i.e., without using an additional linker sequence). In some embodiments, the C-terminus of the first polypeptide is covalently linked to the N-terminus of the second polypeptide by a peptide bond (i.e., without using an additional linker sequence). In some embodiments, the first and second polypeptides are covalently linked by a linker described herein.

[0145] In some embodiments, the inverse monomer contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences of Table 6, Table 8, Table 10, or Table 16.

[0146] In some embodiments, the inverse monomer contains an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences of Table 6, Table 8, Table 10, or Table 16.

[0147] In some embodiments, the inverse monomer is It contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to an hIL10 reverse monomer selected from the group consisting of TIFF2026519565000047.tif17142.

[0148] In some embodiments, the inverse monomer contains an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with an hIL10 inverse monomer selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98.

[0149] (Table 16) Human reverse monomer amino acid sequences TIFF2026519565000048.tif133152

[0150] To facilitate the evaluation of the activity of the reverse monomer of this disclosure in mouse in vivo models of human diseases, this disclosure further provides a mouse reverse IL10 monomer as a substitute for the human reverse IL10 monomer of this disclosure. Preparation of the mouse substitute reverse monomer can be achieved by substitution of the mouse polypeptide with the corresponding human polypeptide substitution according to the following alignment of the mouse sequence and the human sequence (see Figure 11).

[0151] In Figure 11, DR1060_aa / 1-171 has the following amino acid sequence: TIFF2026519565000049.tif24140 contains WC161_aa / 1-172, which has the following amino acid sequence: Includes TIFF2026519565000050.tif24139.

[0152] Inverse monomer dimer In some embodiments, the disclosure provides a polypeptide of formula (1) linked in a dimer format. In some embodiments, the polypeptide of formula (1) is of formula (2): [Monomer 1]-Linker x -[monomer 2](2) The molecule has the structure shown in formula 1, where monomer 1 and monomer 2 are polypeptides of formula 1. In some embodiments, monomer 1 and monomer 2 are the same (i.e., they have the same amino acid sequence). In some embodiments, monomer 1 and monomer 2 are different (i.e., they have different amino acid sequences). In some embodiments, x = 0 (no linker) or 1 (a linker is present).

[0153] In some embodiments, the inverse monomer dimer of formula (2) is formed in the order of amino to carboxyl, (a) Below: (i) A first polypeptide having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), 117-159 (SEQ ID NO: 13), and 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature wild-type human IL-10 (SEQ ID NO: 20), (ii) The first linker where x = 0 (no linker exists) or 1 (a linker exists) x and, (iii) Numbered according to mature wild-type hIL10 (SEQ ID NO:20), amino acid residues 1-160 of human IL-10 (SEQ ID NO:20), amino acid residues 2-160 of human IL-10 (SEQ ID NO:36), amino acid residues 3-160 of human IL-10 (SEQ ID NO:37), amino acid residues 4-160 of human IL-10 (SEQ ID NO:38), amino acid residues 5-160 of human IL-10 (SEQ ID NO:39), amino acid residues 6-160 of human IL-10 (SEQ ID NO:40), amino acid residues 7-160 of human IL-10 (SEQ ID NO:41), amino acid residues 8-160 of human IL-10 (SEQ ID NO:42), amino acid residues 9-160 of human IL-10 (SEQ ID NO: A second polypeptide containing amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 10-160 of human IL-10 (SEQ ID NO:44) and amino acid residues 11-160 of human IL-10 (SEQ ID NO:45), and Includes monomer 1, (b) A second linker where y = 0 (no linker exists) or 1 (a linker exists). y , and (c) Numbered according to mature human IL-10 (SEQ ID NO:20), amino acid residues 1-116 of human IL-10 (SEQ ID NO:15), amino acid residues 2-116 of human IL-10 (SEQ ID NO:16), amino acid residues 3-116 of human IL-10 (SEQ ID NO:17), amino acid residues 4-116 of human IL-10 (SEQ ID NO:18), amino acid residues 5-116 of human IL-10 (SEQ ID NO:19), amino acid residues 6-116 of human IL-10 (SEQ ID NO:29), amino acid residues 7-116 of human IL-10 (SEQ ID NO:30), amino acid residues 8-116 of human IL-10 (SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID Monomer 2) containing a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 10-116 of human IL-10 (SEQ ID NO:33) and amino acid residues 11-116 of human IL-10 (SEQ ID NO:34). Includes.

[0154] In some embodiments, the inverse monomer dimer of formula (2) is formed in the order of amino to carboxyl, (b) Below: (j) A first polypeptide having an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), amino acid residues 117-159 (SEQ ID NO: 13), and amino acid residues 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature wild-type human IL-10 (SEQ ID NO: 20), (ii) The first linker where x = 0 (no linker exists) or 1 (a linker exists) x and, (iii) Numbered according to mature wild-type hIL10 (SEQ ID NO:20), amino acid residues 1-160 of human IL-10 (SEQ ID NO:20), amino acid residues 2-160 of human IL-10 (SEQ ID NO:36), amino acid residues 3-160 of human IL-10 (SEQ ID NO:37), amino acid residues 4-160 of human IL-10 (SEQ ID NO:38), amino acid residues 5-160 of human IL-10 (SEQ ID NO:39), amino acid residues 6-160 of human IL-10 (SEQ ID NO:40), amino acid residues 7-160 of human IL-10 (SEQ ID NO:41), amino acid residues 8-160 of human IL-10 (SEQ ID NO:42), amino acid residues 9-160 of human IL-10 (SEQ ID NO: A second polypeptide containing amino acids having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 10-160 (SEQ ID NO:44) and amino acid residues 11-160 (SEQ ID NO:45) of human IL-10, and Includes monomer 1, (b) A second linker where y = 0 (no linker exists) or 1 (a linker exists). y , and (c) Numbered according to mature human IL-10 (SEQ ID NO:20), amino acid residues 1-116 of human IL-10 (SEQ ID NO:15), amino acid residues 2-116 of human IL-10 (SEQ ID NO:16), amino acid residues 3-116 of human IL-10 (SEQ ID NO:17), amino acid residues 4-116 of human IL-10 (SEQ ID NO:18), amino acid residues 5-116 of human IL-10 (SEQ ID NO:19), amino acid residues 6-116 of human IL-10 (SEQ ID NO:29), amino acid residues 7-116 of human IL-10 (SEQ ID NO:30), amino acid residues 8-116 of human IL-10 (SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID Monomer 2) containing a polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 10-116 of human IL-10 (SEQ ID NO:33) and amino acid residues 11-116 of human IL-10 (SEQ ID NO:34). Includes.

[0155] In some embodiments of the inverse monomer of equation (2), the first linker x This is the second linker y It is independent of the second linker. y This is different. In some embodiments, the inverse monomer of equation (2) is x=0 (no linker) or 1 (a linker exists). In some embodiments, y=0 (no linker) or 1 (a linker exists). In some embodiments, the first linker x and the second linker yIf present, they may be the same or different. In some embodiments, there is no linker between the first polypeptide and the second polypeptide of monomer 1, and / or between monomer 1 and monomer 2 of formula (2). In some embodiments, the first linker (ii) (e.g., linker x ) is present between the first polypeptide and the second polypeptide of monomer 1, and / or the second linker (b) (e.g., linker y ) is present between monomer 1 and monomer 2 of formula (2). In some embodiments, the first and second linkers include an amino acid sequence of 1, 2, 3, 4, or 5 amino acids.

[0156] Exemplary embodiments of the polypeptide of formula (2) are provided in Table 13 below.

[0157] (Table 13) Exemplary dimers and inverse monomers TIFF2026519565000051.tif252139

[0158] In some embodiments, the inverse hIL10 monomer contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an hIL10 inverse monomer selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51.

[0159] In some embodiments, the inverse hIL10 monomer contains an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with an hIL10 inverse monomer selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51.

[0160] In some embodiments, the inverse monomer dimer of formula (2) is formed in the order of amino to carboxyl, (a) Below: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), 117-159 (SEQ ID NO: 13), and 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature human IL-10 (SEQ ID NO: 20), (ii) First linker x , (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1-116 (SEQ ID NO: 15) of human IL-10. Includes monomer 1, (b) Second linker y , (c) Below: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 (SEQ ID NO: 12), 117-159 (SEQ ID NO: 13), and 117-160 (SEQ ID NO: 14) of human IL-10, numbered according to mature human IL-10 (SEQ ID NO: 20), (ii) Third linkerz and (iii) a second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1 to 116 (SEQ ID NO: 15) of human IL-10 comprising monomer 2 comprising

[0161] In some embodiments, the first, second and third linkers are independent of each other and different from each other. In some embodiments, x, y and / or z = 0 (no linker) or 1 (linker is present). In some embodiments, the first, second and / or third linkers (e.g., linker x , linker y and linker z ) if present, may be the same or different independently of each other. For example, in some embodiments, the first, second and / or third linkers are the same (e.g., comprising the same amino acid sequence). In some embodiments, the first, second and / or third linkers are different (e.g., each linker comprises a different amino acid sequence or one linker comprises an amino acid sequence different from the other two linkers). In some embodiments, there is no linker between the first polypeptide and the second polypeptide of monomer 1, between monomer 1 and monomer 2, or between the first polypeptide and the second polypeptide of monomer 2 of formula (2). In some embodiments, the first linker (e.g., linker x ) is present between the first polypeptide and the second polypeptide of monomer 1. In some embodiments, the second linker (e.g., linker y ) is present between monomer 1 and monomer 2 of formula (2). In some embodiments, the third linker (e.g., linker z ) is present between the first polypeptide and the second polypeptide of monomer 2. In some embodiments, the first, second and / or third linkers comprise an amino acid sequence of 1, 2, 3, 4 or 5 amino acids.

[0162] In any of the aspects of the present disclosure, the reverse monomer or reverse monomer dimer polypeptide can further optionally include a polypeptide sequence useful for polypeptide purification at the carboxy terminus (-COOH). In some aspects, the sequence at the carboxy terminus (-COOH) is GGS-8xHis TIFF2026519565000052.tif4128.

[0163] In some aspects, the reverse monomer dimer includes the amino acid sequence of Table 8, Table 12, Table 13 or Table 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of Table 8, Table 12, Table 13 or Table 16.

[0164] In some aspects, the reverse monomer dimer includes the amino acid sequence of Table 8, Table 12, Table 13 or Table 16, or an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity to the amino acid sequence of Table 8, Table 12, Table 13 or Table 16.

[0165] Reverse monomer variant This disclosure provides polypeptides having one or more (e.g., one, two, three, four, five, or all six) amino acid substitutions compared to the reference sequences of domains [A], [B], [C], [D], [E], and [F], in addition to the reference sequences of domains [A], [B], [C], [D], [E], and [F] (including amino acid sequences derived from the wild-type hIL10 sequence). The reference sequences of domains [A], [B], [C], [D], [E], and [F] are primarily composed of alpha-helical domains. As used herein, the term “helix” is as defined in the RCSB Protein Data Bank, entry 2ILK, version 1.2 (see www.rcsb.org / sequence / 2ILK on the internet). Accordingly, in some embodiments of this disclosure, amino acid substitutions in one or more of the domains [A], [B], [C], [D], [E], and [F] of the polypeptide of formula 1 have amino acid substitutions that are favorable for alpha-helix formation (and / or do not contribute to alpha-helix destabilization). In some embodiments, amino acid substitutions at one or more given positions in the domains [A], [B], [C], [D], [E], and [F] of the polypeptide of formula 1 result in a minimum free energy difference (Δ(ΔG)) compared to amino acids at the same position in the reference sequence. In some embodiments, the amino acids substituted in one or more of the domains [A], [B], [C], [D], [E], and [F] are selected from the group consisting of methionine (M), alanine (A), leucine (L), glutamic acid (E), and lysine (K). In some embodiments, the polypeptide of formula 1 contains one or two amino acid substitutions compared to one or more reference sequences from domains [A], [B], [C], [D], [E], and [F], provided that the amino acid substitutions are not one or more amino acids selected from the group consisting of glycine (G) and proline (P).

[0166] In some embodiments, domain [C] includes a substitution from D to K at position 8 of SEQ ID NO:3. In some embodiments, domain [F] includes a substitution from T to L at position 14 of SEQ ID NO:6.

[0167] In some embodiments, the disclosure provides a variant inverse monomer or dimer comprising one or more amino acid substitutions at positions corresponding to residues of mature human IL10 or mature mouse IL10. In some embodiments, the inverse monomer or dimer comprises one or more amino acid substitutions at positions corresponding to residues D25 or T100 of the mature human IL10 sequence or mature mouse IL10 sequence. In some embodiments, the variant inverse monomer comprises amino acid substitution D25K numbered according to the mature human IL10 sequence or mature mouse IL10 sequence. In some embodiments, the inverse monomer comprises the following amino acid sequence: Contains a sequence that has at least 95% sequence identity with TIFF2026519565000053.tif24128 or SEQ ID NO:53.

[0168] In some embodiments, the amino acid substitution at the residue corresponding to T100 is T100L.

[0169] In some embodiments, the variant reverse monomer contains amino acid substitutions T100L numbered according to a mature human IL10 sequence or a mature mouse IL10 sequence. In some embodiments, the reverse monomer has the following amino acid sequence: Contains a sequence that has at least 95% sequence identity with TIFF2026519565000054.tif24128 or SEQ ID NO:91.

[0170] In some embodiments, one inverse monomer of the dimerized inverse monomer contains either or both of the amino acid substitution (numbered according to the mature human IL10 sequence or the mature mouse IL10 sequence) at position D25 or T100. In some embodiments, each inverse monomer of the dimerized inverse monomer contains the amino acid substitution at either or both of the positions D25 or T100 (numbered according to the mature human IL10 sequence or the mature mouse IL10 sequence). In some embodiments, one inverse monomer of the dimerized inverse monomer contains the amino acid substitution D25K (numbered according to the mature human IL10 sequence or the mature mouse IL10 sequence). In some embodiments, one inverse monomer of the dimerized inverse monomer contains the amino acid substitution T100L (numbered according to the mature human IL10 sequence or the mature mouse IL10 sequence). In some embodiments, each reverse monomer of the dimerized reverse monomer contains one or both of the amino acid substitution (numbered according to the mature human IL10 sequence or the mature mouse IL10 sequence) at position D25 or T100. In some embodiments, the variant reverse monomer contains the amino acid substitution D25K, numbered according to the mature human IL10 sequence or the mature mouse IL10 sequence. In some embodiments, the reverse monomer dimer has the following amino acid sequence: Contains a sequence that has at least 95% sequence identity with TIFF2026519565000055.tif44128 or SEQ ID NO:54.

[0171] In some embodiments, the amino acid substitution at the residue corresponding to T100 in the inverse monomeric dimer is T100L. In some embodiments, the inverse monomeric dimer has the following amino acid sequence: Contains a sequence that has at least 95% sequence identity with TIFF2026519565000056.tif44128 or SEQ ID NO:55.

[0172] Biased activity An inflammatory response is a series of biological events in mammals initiated in response to infectious and / or damaging stimuli that mitigate the possibility of systemic infection. Typically, mammalian inflammatory responses are mediated by myeloid cells, particularly macrophages activated by exogenous stimuli, such as components of bacterial cell walls, e.g., lipopolysaccharides ("LPS") of Gram-negative bacteria. Activated myeloid cells act as precursors to infection and / or injury by secreting a variety of pro-inflammatory signaling molecules, including, but not limited to, interleukin-6 (IL-6), interleukin-1 (IL-1, particularly IL-1β), and tumor necrosis factor alpha (TNFα), which initiate and / or mediate multiple biological processes associated with the inflammatory response.

[0173] While the inflammatory response is essential for protecting mammalian subjects from infection, excessive and / or chronic activation of immune cells, particularly myeloid cells, is associated with tissue damage, organ dysfunction, and autoimmune diseases. A wide variety of human diseases, without limitation, are associated with excessive and / or chronic inflammation, including inflammatory bowel disease (IBD), rheumatoid arthritis (RA), Alzheimer's disease, asthma, type 1 and type 2 diabetes, and cancer. Subjects produce certain molecules, e.g., IL-10, that act (in addition to other activities) to attenuate the inflammatory response and prevent adverse effects associated with excessive inflammation. Genetic loss of IL-10 in both mice and humans is associated with severe inflammatory bowel disease (IBD). IL-10 expression and secretion correlate, without limitation, with suppression of the inflammatory response in immune cells, including inhibition of the expression and / or secretion of pro-inflammatory cytokines, as well as antigen presentation by activated myeloid cells. IL-10 is also associated with pro-inflammatory activity, particularly in activated CD8+ T cells, despite its central role in suppressing the inflammatory response. Contact between activated CD8+ T cells and IL-10 has been observed to result in enhanced secretion of the pro-inflammatory cytokine interferon-gamma (IFNγ), as well as the release of cytolytic factors such as granzyme A and granzyme B. These competing pro-inflammatory and anti-inflammatory effects present challenges to the therapeutic use of IL-10 in treating inflammatory diseases in mammals.

[0174] In some embodiments, the reverse IL10 monomer is a biased IL10 partial agonist comprising a polypeptide of formula (1) or formula (2) that exhibits cell-type biased activity compared to the wild-type IL10 species from which the reverse IL10 monomer is derived. As used herein, the term “biased” as used in relation to the IL10 reverse monomer (and / or its dimer) is used to indicate that the biased IL10 reverse monomer (or its dimer) exhibits a percentage level of wild-type IL10 activity in a first cell type that is greater than the level of wild-type IL10 activity in a second cell type compared to the wild-type IL10 species from which the reverse IL10 monomer is derived. In one embodiment, the first cell type is cells of myeloid origin, including myeloid cells. In some embodiments, myeloid cells are selected from myelocytes, granulocytes (e.g., neutrophils, eosinophils, or basophils), mast cells, or monocytes. In some embodiments, monocytes are macrophages or dendritic cells. In some embodiments, macrophages are Kupffer cells. In one embodiment, the first cell type is activated myeloid cells. In another embodiment, the first cell type is LPS-activated human myeloid cells. In some embodiments, the second cell type is T cells.

[0175] The inverse IL10 monomers and / or dimers of this disclosure can inhibit pro-inflammatory responses and / or STAT3-mediated signaling in a cell-type-dependent manner, such that inflammatory macrophage activation is inhibited without substantially promoting the production of inflammatory cytokines such as interferon-γ by T cells. In some embodiments, the inverse monomers and / or dimers of this disclosure retain the immunosuppressive function of wild-type hIL10, e.g., inhibiting the production of inflammatory cytokines, while retaining the immunostimulatory function of wild-type hIL10, e.g., CD8 +It reduces IFN-gamma production by T cells. For example, in some embodiments, the inverse monomer and / or dimer of the Disclosure retain activity equivalent to wild-type hIL10 to suppress myeloid cell activation (e.g., as assessed by increased STAT3-mediated signaling in myeloid cells), but substantially reduces activation in PBMCs, T cells, B cells, and NK cells (e.g., as assessed by reduced IFN-gamma production).

[0176] In some embodiments, the inverse monomer and / or dimer of the present disclosure are hIL10 partial agonists.

[0177] Pro-inflammatory activity and anti-inflammatory activity In some embodiments, a reverse IL10 monomer comprising a polypeptide of formula (1) or formula (2) is a biased IL10 partial agonist exhibiting (a) a significant level of at least one anti-inflammatory property of wild-type IL10 and (b) a significantly reduced level of at least one pro-inflammatory property of wild-type IL10. In some embodiments, “significant level of at least one anti-inflammatory property” means that the Emax of the biased IL10 reverse monomer with respect to such anti-inflammatory property is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the Emax level of such anti-inflammatory property exhibited by wild-type IL10, when determined in the test system. Examples of anti-inflammatory properties that can be measured in the test system include, but are not limited to, (a) suppression of hILβ expression and / or secretion by activated human myeloid cells, (b) suppression of hIL6 expression and / or secretion by activated human myeloid cells, and (c) suppression of hTNFα expression and / or secretion by activated human myeloid cells. In some embodiments, activated human myeloid cells are obtained by isolating human monocytes from the buffy coat of a centrifuged anticoagulant-treated human blood sample according to a procedure well known in the art, and activating the isolated monocytes by contacting them with lipopolysaccharide [LPS]. The levels of hILβ, hIL6, and hTNFα expressed and / or secreted by activated monocytes can be determined by immunoassay or flow cytometry according to a procedure well known in the art. One protocol for evaluating the suppression of hILβ, hIL6, and hTNF expression and / or secretion by LPS-activated human monocytes is provided in the examples herein.

[0178] In some embodiments, “a markedly reduced level of at least one pro-inflammatory property” means that the Emax of the biased IL10 inverse monomer with respect to such pro-inflammatory property is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the Emax of wild-type IL10 for that pro-inflammatory property, if determined in the test system. Examples of pro-inflammatory properties include, but are not limited to, (a) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (b) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (c) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells. In some embodiments, activated human T cells are obtained by isolating human whole blood CD8+ T cells, and the isolated CD8+ cells are activated by contacting them with anti-CD3 and anti-CD28 antibodies according to procedures well known in the art. The levels of IFNγ, granzyme A, and granzyme B expressed and / or secreted by the isolated CD8+ T cells can be determined by immunoassay or flow cytometry according to procedures well known in the art. One protocol for evaluating the expression and / or secretion of IFNγ, granzyme A, and granzyme B expressed and / or secreted by CD3 / CD28 activated CD8+ T cells is provided in the following examples.

[0179] In some embodiments, a reverse IL10 monomer comprising a polypeptide of formula (1) or formula (2) is a biased hIL10 partial agonist exhibiting a marked level of at least one anti-inflammatory property of wild-type hIL10 and a markedly reduced level of at least one pro-inflammatory property of wild-type IL10, wherein (a) the marked level of at least one anti-inflammatory property of wild-type hIL10 is the Emax of at least one anti-inflammatory property exceeding 30% of the Emax of such anti-inflammatory property exhibited by wild-type hIL10, and the at least one anti-inflammatory property is (i) suppression of hILb expression and / or secretion in LPS-activated human monocytes, and (ii) suppression of hIL6 expression and / or secretion in LPS-activated human monocytes. (iii) Suppression of hTNFα expression and / or secretion in LPS-activated human monocytes, and (b) a markedly reduced level of at least one pro-inflammatory property of wild-type hIL10 is the Emax of at least one anti-inflammatory property less than 30% of the Emax of such anti-inflammatory property exhibited by wild-type hIL10, and the at least one pro-inflammatory property is selected from the group consisting of (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells.

[0180] In some embodiments, the reverse IL10 monomer of the present disclosure comprising the polypeptide of formula (1) or formula (2) is a biased hIL10 partial agonist that exhibits a significant level of at least one anti-inflammatory property of wild-type hIL10 and a significantly reduced level of at least one pro-inflammatory property of wild-type IL10, wherein the significant level of at least one anti-inflammatory property of wild-type hIL10 is an Emax of at least one anti-inflammatory property that exceeds 30% of the Emax of such anti-inflammatory properties exhibited by wild-type hIL10, and the at least one anti-inflammatory property is selected from the group consisting of (i) suppression of the expression and / or secretion of hILb in LPS-activated human monocytes, (ii) suppression of the expression and / or secretion of hIL6 in LPS-activated human monocytes, or (iii) suppression of the expression and / or secretion of hTNFa in LPS-activated human monocytes, and (b) the significantly reduced level of at least one pro-inflammatory property of wild-type hIL10 is an Emax of at least one anti-inflammatory property that is less than 30% of the Emax of such anti-inflammatory properties exhibited by wild-type hIL10, and the at least one pro-inflammatory property is selected from the group consisting of (i) suppression of the expression and / or secretion of IFNγ by activated human CD8+ T cells, (i) suppression of the expression and / or secretion of granzyme A by activated human CD8+ T cells, and (iii) suppression of the expression and / or secretion of granzyme A by activated human CD8+ T cells.

[0181] In some embodiments, the reverse IL10 monomer of the Disclosure, comprising a polypeptide of formula (1) or formula (2), is a biased reverse hIL10 monomer, and in an assay of anti-inflammatory activity selected from the group consisting of (a)(i) suppression of hILβ expression and / or secretion in LPS-activated human monocytes, (ii) suppression of hIL6 expression and / or secretion in LPS-activated human monocytes, or (iii) suppression of hTNFα expression and / or secretion in LPS-activated human monocytes, wild-type hIL10 In assays for pro-inflammatory activity, the Emax of the reverse hIL10 monomer is greater than 30% of the Emax of wild-type hIL10. This is selected from a group consisting of (b) (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (i) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells. In these assays, the Emax of the reverse hIL10 monomer is less than 10% of the Emax of wild-type hIL10.

[0182] In some embodiments, the reverse IL10 monomer of the Disclosure, comprising a polypeptide of formula (1) or formula (2), is a biased reverse hIL10 monomer, and in an assay of anti-inflammatory activity selected from the group consisting of (a)(i) suppression of hILβ expression and / or secretion in LPS-activated human monocytes, (ii) suppression of hIL6 expression and / or secretion in LPS-activated human monocytes, or (iii) suppression of hTNFα expression and / or secretion in LPS-activated human monocytes, wild-type hIL10 In assays for pro-inflammatory activity, the Emax of the reverse hIL10 monomer is greater than 50% of the Emax of wild-type hIL10. This includes (b) (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (i) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells.

[0183] In some embodiments, the reverse IL10 monomer of the Disclosure, comprising a polypeptide of formula (1) or formula (2), is a biased reverse hIL10 monomer, and in an assay of anti-inflammatory activity selected from the group consisting of (a)(i) suppression of hILβ expression and / or secretion in LPS-activated human monocytes, (ii) suppression of hIL6 expression and / or secretion in LPS-activated human monocytes, or (iii) suppression of hTNFα expression and / or secretion in LPS-activated human monocytes, wild-type hIL10 In assays for pro-inflammatory activity, the Emax of the reverse hIL10 monomer is greater than 50% of the Emax of wild-type hIL10. This is selected from a group consisting of (b) (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (i) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells, and (iii) suppression of granzyme A expression and / or secretion by activated human CD8+ T cells. In these assays, the Emax of the reverse hIL10 monomer is less than 10% of the Emax of wild-type hIL10.

[0184] STAT3 As mentioned above, the interaction between IL10 and the IL10 receptor results in intracellular signaling characterized by enhanced intracellular production of phosphorylated STAT3 (phospho-STAT3). Consequently, one measure of IL10 activity that can be assessed using cells expressing the IL10 receptor (composed of IL10Ra and IL10Rb) is intracellular production of phospho-STAT3.

[0185] In one embodiment, the polypeptide of formula (1) or formula (2) is a biased hIL10 partial agonist, the first cell type is activated human myeloid cells, the second cell type is activated human T cells, and the level of IL10 activity is measured by intracellular production of phospho-STAT3. In another embodiment, the polypeptide of formula (1) or formula (2) is a biased hIL10 partial agonist that maintains a higher proportion of hIL10 activity relative to activated human monocytes than activated human CD8+ T cells, and the level of IL10 activity is measured by intracellular production of phospho-STAT3. In several embodiments, the levels of IL10 activity in the first and second cell types are measured by the production of phospho-STAT3 in the first cell type in response to contacting the first and second cell types with a reverse IL10 monomer containing the polypeptide of formula (1) or formula (2).

[0186] In some embodiments, the relative activation of STAT3 signaling by the inverse monomer and / or dimer described herein in a first cell type versus a second cell type differs from the relative activation of STAT3 signaling by wild-type human IL10 or wild-type mouse IL10 in a first cell type versus a second cell type. In some embodiments, the level of intracellular phospho-STAT3 induced in human myeloid cells in response to contact with a sufficient amount of human IL10 inverse monomer (or dimer) is at least 10 times, or at least 100 times, or at least 1000 times, the level of intracellular phospho-STAT3 induced in human lymphocyte cells in response to contact with the same amount of human IL10 inverse monomer (or dimer). In one embodiment, the ratio of the level of STAT3 signaling induced in myeloid cells in response to contact with human IL-10 reciprocal monomer to the level of STAT3 signaling induced in lymphocytes in response to contact with human IL-10 reciprocal monomer is different from (greater than or less than) the ratio of the level of STAT3 signaling induced in myeloid cells in response to contact with wild-type hIL-10 to the level of STAT3 signaling induced within lymphocytes in response to contact with wild-type hIL-10. In some embodiments, the ratio of the activity of human IL-10 reciprocal monomer (and / or its dimer) in human myeloid cells compared to human lymphocytes (as determined by the level of intracellular phospho-STAT3) is greater than the ratio of the activity of wild-type human IL-10 in human myeloid cells compared to human lymphocytes. In some embodiments, myeloid cells are neutrophils, eosinophils, mast cells, basophils, or monocytes. In some embodiments, monocytes are macrophages or dendritic cells. In some embodiments, macrophages are Kupffer cells. In some embodiments, lymphocytes are CD8+ T cells, CD4+ T cells, B cells, or NK cells.

[0187] In some embodiments, the inverse monomer and / or dimer of the present disclosure are used in bone marrow cells to control the pSTAT3 E of wild-type hIL10. maxpENTER3 E max (See, for example, Figure 2A). In some embodiments, the inverse monomer and / or dimer of the Disclosure exhibit reduced STAT3-mediated signaling in lymphocytes such as T cells, B cells, or NK cells compared to wild-type hIL10 (See, for example, Figure 2B). In some embodiments, the inverse monomer and / or dimer of the Disclosure exhibit reduced pSTAT3 E in lymphocytes compared to wild-type hIL10. max pSTAT3 E less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% max It is present in lymphocytes. In some embodiments, the inverse monomer and / or its dimer are present in lymphocytes as pSTAT3 E of the wild-type or parental IL10 polypeptide. max Less than 70% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, or less than 30%) but more than 20% of pSTAT3 E max This is brought about in lymphocytes. In some embodiments, the lymphocytes are selected from CD8+ T cells, CD4+ T cells, B cells, or NK cells.

[0188] Linker In some embodiments, the disclosure provides polypeptide linkers that can be used to covalently bond functional subunits of a polypeptide, which are composed of a plurality of functional domains or functional subunits. The linkers are typically long enough to allow some movement between the functional domains or functional subunits of the polypeptide to which they bond. In some embodiments, the linker is located between one or more different helices or domains [A], [B], [C], [D], [E], and [F] of formula (1). In some embodiments, the linker is located between two monomers of formula (2).

[0189] The linker can be readily selected and may be of any suitable length, e.g., 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids. Examples of linkers useful for the implementation of this disclosure include, but are not limited to, glycine polymers (G)n (SEQ ID NO: 138) where is an integer from 1 to 50. Examples of linkers useful for the implementation of this disclosure include, but are not limited to, glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers, also referred to herein as "GS-linkers." Because glycine polymers and glycine-serine polymers are relatively unstructured, they can function as flexible tethers between domains or subunits of polypeptides. In some embodiments, the GS-linker is of the formula: TIFF2026519565000057.tif31161 and selected polymers of combinations thereof, where m, n, and o are independently selected integers from 1 to 20, e.g., 1 to 18, 2 to 16, 3 to 14, 4 to 12, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. Examples of GS-linkers include, without limitation, Examples include TIFF2026519565000058.tif37152 and its polymers.

[0190] Modifications to provide additional functionality In some embodiments, the inverse monomer and / or its dimer may include a functional domain of a chimeric polypeptide. The inverse monomer fusion proteins of this disclosure can be readily produced by recombinant DNA methods by techniques known in the art, by constructing a recombinant vector comprising a nucleic acid sequence comprising a nucleic acid sequence encoding a fusion partner at either the N-terminus or C-terminus of the inverse monomer and a nucleic acid sequence encoding the inverse monomer in frame, wherein the sequence optionally further comprises a nucleic acid sequence encoding a linker polypeptide or spacer polypeptide in frame.

[0191] In other embodiments, the inverse monomer and / or dimer may be modified to include an additional polypeptide sequence that functions as an antigen tag, such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated, highly specific anti-FLAG antibody, as described herein (see, e.g., Blanar et al. (1992) Science 256:1014 and LeClair, et al. (1992) PNAS-USA 89:8145). In some embodiments, the binding molecule further includes a C-terminal c-myc epitope tag.

[0192] In some embodiments, inverse monomers and / or dimers that optionally incorporate a 1-40 (or 2-20, or 5-20, or 10-20) amino acid linker molecule between the inverse monomer sequence and the sequence of the targeting domain of the fusion protein are conjugated to the molecule ("targeting domain") to facilitate selective binding to specific cell types or tissues that express cell surface molecules that specifically bind to such targeting domain.

[0193] In other embodiments, a chimeric polypeptide comprising an inverse monomer and an antibody or its antigen-binding moiety can be generated. The antibody or antigen-binding component of the chimeric protein may serve as a targeting site. For example, a chimeric polypeptide may be used to localize a chimeric protein to a specific subset of cells or target molecules. In some embodiments, the targeting domain is an antibody. As used herein, the term “antibody” means any form of antibody (also known as immunoglobulin (Ig)) that exhibits desired biological activity to bind to an antigen epitope, as described herein. The term “antibody” as described herein, without limitation, includes polyclonal antibodies, monoclonal antibodies (including full-length monoclonal antibodies containing two light chains and two heavy chains), multispecific antibodies (e.g., bispecific antibodies that bind to two or more antigens or antigenic epitopes on a single antigen), fully human antibodies (huAb), humanized antibodies (hzAb), chimeric antibodies, single-chain variable fragment antibodies (scFv), single-domain antibodies (sdAb), variable weight (VH) domain antibodies, diabolic (dAb), and antigen-binding fragments of heavy-chain-only antibodies (VHH) containing the amino acid sequence of a variable region. As used herein, the term “antibody” means (a) glycosylated and nonglycosylated immunoglobulins (including, but not limited to, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4) that specifically bind to target molecules such as antigens, and (b) IgG(1-4) delta C2 immunoglobulins that compete for binding to target molecules with the immunoglobulin from which they originate, but not limited to those immunoglobulins. H 2, F(ab')2, Fab, ScFv, V H , V LThe term antibody collectively refers to immunoglobulin derivatives including tetrabodies, triabodies, diabodies, dsFv, F(ab')3, scFv-Fc, and (scFv)2. The term antibody is not limited to immunoglobulins derived from any specific mammalian species, but includes mouse antibodies, human antibodies, horse antibodies, camelid antibodies, and uman antibodies. The term antibody includes "heavy chain antibodies," "VHH," and "nanobodies" typically obtained from immunization, including camelids (camels, llamas, and alpacas), as described in more detail below in the definition of "VHH," for example, as described in Hamers-Casterman et al. 1993. Nature. 363:446-448. The term “antibody” encompasses antibodies that can be isolated from natural sources or from animals after antigen-mediated immunization, and engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR transplant antibodies, veneered antibodies, or deimmunized antibodies (e.g., for removing B and / or T cell epitopes). In some embodiments, the targeting domain specifically binds to cell surface markers of pro-inflammatory cells, such as activated immune cells. In some embodiments, the targeting domain is an antibody that selectively binds to cell surface markers, including, but not limited to, IL1R1 receptor, IL-1 receptor accessory protein, IL6 receptor subunit (IL6R), HLA-DR, HLA-DR α chain, HLA-DR β chain, TNFR1, TNFR2, CD4, CD8, F4 / 80, CCR2, CD169, CX3CR1, CD206, CD163, or Lyve1. Methods for generating cytokine-antibody chimeric polypeptides are described, for example, in U.S. Patent No. 6,617,135.

[0194] Association with carrier molecules to increase the duration of action The inverse monomers and / or dimers described herein may be modified to provide in vivo lifetime extension and / or duration of action in a target. In some embodiments, the inverse monomers and / or dimers are conjugated to a carrier molecule to provide a desired pharmacological property, such as an extension of half-life. In some embodiments, the inverse monomers and / or dimers are covalently bound to the Fc domain of IgG, albumin, a water-soluble polymer, or other molecule to extend their half-life, such as glycosylation, acylation, etc., as known in the art. In some embodiments, inverse monomers and / or dimers modified to provide an extended duration of action in mammalian subjects have a half-life in mammalian subjects of more than 4 hours, or more than 5 hours, or more than 6 hours, or more than 7 hours, or more than 8 hours, or more than 9 hours, or more than 10 hours, or more than 12 hours, or more than 18 hours, or more than 24 hours, or more than 2 days, or more than 3 days, or more than 4 days, or more than 5 days, or more than 6 days, or more than 7 days, or more than 10 days, or more than 14 days, or more than 21 days, or more than 30 days.

[0195] Modifications of inverse monomers and / or dimers to provide an extended duration of action in mammalian subjects include (without limiting) the following: • Conjugation of inverse monomers to one or more protein carrier molecules, Optionally, conjugation of the inverse monomer to a protein carrier molecule in the form of a fusion protein having an additional polypeptide sequence (e.g., an inverse monomer-Fc fusion), and Conjugation to polymers (e.g., water-soluble polymers for providing PEGylated IL10 reverse monomer polypeptides).

[0196] It should be noted that several types of modifications that provide an extension of the duration of action in mammalian subjects may be used with respect to a given inverse monomer and / or dimer. For example, the hIL10 inverse monomer and / or dimer of this disclosure may include amino acid substitutions that provide an extension of the duration of action and conjugation with a carrier molecule such as a polyethylene glycol (PEG) molecule.

[0197] Protein carrier molecules: Examples of protein carrier molecules that can be covalently bonded to inverse monomers and / or dimers to provide an extension of the duration of action in vivo include, but are not limited to, albumin, antibodies, and antibody fragments, such as the Fc domain of an IgG molecule.

[0198] Fc fusion: In some embodiments, the inverse monomer of this disclosure is conjugated to an Fc domain. Fc fusion conjugates have been shown to increase the systemic half-life of biologics, thus potentially reducing the administration frequency required for biologic products. Fc binds to neonatal Fc receptors (FcRn) in endothelial cells lining blood vessels. Upon binding, the Fc fusion molecule is protected from degradation and re-released in circulation, allowing it to circulate for an even longer period. This Fc binding is thought to be the mechanism by which endogenous IgG maintains its long plasma half-life. Furthermore, recent Fc fusion technologies ligate a single copy of a biologic to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biologic compared to conventional Fc fusion conjugates. A “Fc region” useful for the preparation of Fc fusions can be a natural or synthetic polypeptide homologous to the IgG C-terminal domain produced by papain digestion of IgG. IgG Fc has a molecular weight of approximately 50 kDa. The conjugating molecules described herein may be conjugated to the entire Fc region or to a smaller portion that retains the ability to extend the cyclic half-life of the chimeric polypeptide, of which it is a part. Furthermore, the full-length Fc region or the fragmented Fc region may be variants of the wild-type molecule. In a typical presentation, each monomer of the dimeric Fc may have heterologous polypeptides, which may be the same or different.

[0199] As shown, the linking of the inverse monomer to the Fc subunit may involve the incorporation of a linker molecule between the inverse monomer and the Fc subunit. In some embodiments, the inverse monomer is expressed as a fusion protein with an Fc domain incorporating the amino acid sequence of the hinge region of an IgG antibody. The Fc domain manipulated according to the above may be derived from the mammalian IgG species IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain may be derived from the human IgG1, IgG2, IgG3, and IgG4 IgG species. In some embodiments, the hinge region is the hinge region of IgG1. In a particular embodiment, the inverse monomer is linked to the Fc domain using the human IgG1 hinge domain.

[0200] In some embodiments, the linker is a chemical linker. Examples of chemical linkers include arylacetylenes, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacitors, amino acids, or combinations thereof. In some embodiments, the linker is a peptide linker. Peptide linkers may contain 1 to 50 amino acids (e.g., 2 to 50, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 5 amino acids). Glycine polymers and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between components. Examples of glycine polymers include (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., (GmSo)n (SEQ ID NO: 136), (GSGGS)n (SEQ ID NO: 56), (GmSoGm)n (SEQ ID NO: 59), (GmSoGmSoGm)n (SEQ ID NO: 60), (GSGGSm)n (SEQ ID NO: 61), (GSGSmG)n (SEQ ID NO: 62), and (GGGSm)n (SEQ ID NO: 63), as well as combinations thereof, where m, n, and o are integers at least 1 to 20, e.g., 1 to 18, 216, 3 to 14, 4 to 12, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively), and other flexible linkers.

[0201] In some embodiments, the amino acid sequence of the Fc domain is modified to reduce effector function. In some embodiments, the Fc domain may be modified to substantially reduce binding to Fc receptors (FcyR and FcR), thereby reducing or eliminating antibody-directed cytotoxicity (ADCC) effector function. Modifications of the Fc domain to reduce effector function are well known in the art. See, for example, Wang, et al. (2018) IgG Fc engineering to modulate antibody effector functions, Protein Cell 9(1):63-73. For example, it is known that changing the lysine residue at position 235 (EU numbering) from leucine (L) to glutamic acid (E) reduces effector function by decreasing the binding of FcgR to C1q. Alegre, et al. (1992) J. Immunology 148:3461-3468. In addition, substitutions of two leucine (L) residues at positions 234 and 235 (EU numbering) within the IgG1 hinge region by alanine (A), namely L234A and L235A, reduce complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Hezereh et al., (2001) J. Virol 75(24):12161-68. Furthermore, mutations of proline at position 329 (EU numbering) to alanine (P329A) or glycine (P329G) may mitigate effector function and may be combined with L234A and L235A substitutions. In some embodiments, the Fc domain may contain the amino acid substitution L234A / L235A / P329A (EU numbering), known as the "LALAPA" substitution, or L234A / L235A / P329G (EU numbering), known as the "LALAPG" substitution. In some embodiments, the Fc domain may contain the amino acid substitution E233P / L234V / L235A / ΔG237 (known in scientific literature as the PVAdelG mutation).

[0202] In some embodiments, the Fc domain is derived from hIgG4. In IgG4, glycosylation at position 297 (EU numbering) has been shown to contribute to effector function. Edelman, et al (1969) PNAS (USA) 63:78-85. Examples of modifications at N297 to eliminate the glycosylation site and effector function within the Fc domain of hIgG4 include amino acid substitutions selected from N297Q and N297G (EU numbering).

[0203] In some embodiments, the amino acid sequence of the Fc domain is modified to incorporate amino acid substitutions that extend the duration of the molecule's action and prevent clearance. In some embodiments, such modifications to the Fc domain include amino acid substitutions M428L and N434S (EU numbering), referred to as "LS" modifications. LS modifications may be optionally combined with amino acid substitutions to reduce effector function.

[0204] In some embodiments, the amino acid sequence of the Fc domain can be further modified to eliminate either an N-linked or O-linked glycosylation site. In particular, deglycosylation variants of the Fc domain of IgG1 subclasses are known to have poor mediators of effector function. (Jefferies et al. 1998, Immol. Rev., vol. 163, 50-76).

[0205] In some embodiments, the inverse monomer or dimer of the present disclosure may be further modified to extend its duration of action in vivo. In some embodiments, conjugation of the PEG moiety may be achieved via a sulfhydryl (-SH) group of a cysteine ​​residue. In some embodiments, PEGylation of the inverse monomer / Fc fusion polypeptide is provided to a naturally occurring cysteine ​​residue at position 220 (C220, EU numbering) of the upper hinge region of the Fc domain.

[0206] Albumin carrier molecule In some embodiments, the inverse monomer and / or its dimer are conjugated to albumin molecules known in the art (e.g., human serum albumin) to facilitate the extension of in vivo exposure. In some embodiments, the inverse monomer and / or its dimer are conjugated to albumin via a chemical bond or expressed as a fusion protein with an albumin molecule (referred herein to as “inverse monomer-albumin fusions”). The term “albumin” as used in relation to inverse monomer-albumin fusions includes albumins such as human serum albumin (HSA), cynomolgus monkey serum albumin, and bovine serum albumin (BSA). In some embodiments, HSA contains a C34S or K573P amino acid substitution compared to the wild-type HSA sequence. According to this disclosure, albumin can be conjugated to a carboxyl-terminated, amino-terminated, or both carboxyl-terminated and amino-terminated, as well as to an inverse monomer internally (see, for example, U.S. Patent Nos. 5,876,969 and 7,056,701). In the inverse il10 monomer HSA fusions contemplated by this disclosure, various forms of albumin can be used, such as albumin secretory presequences and their variants, fragments and variants thereof, and HSA variants. Such forms generally have one or more desired albumin activities. In additional embodiments, this disclosure involves a fusion protein comprising an inverse monomer directly or indirectly fused to albumin, albumin fragments and albumin variants, etc., wherein the fusion protein has higher plasma stability than the unfused drug molecule, and / or the fusion protein retains the therapeutic activity of the unfused drug molecule. As an alternative to the chemical bond between the inverse monomer and the albumin molecule, the inverse monomer-albumin complex may be provided as a fusion protein comprising an albumin polypeptide sequence and an inverse monomer recombinantly expressed in a host cell as a single polypeptide chain, optionally including a linker molecule between albumin and the inverse monomer. Such fusion proteins can be readily prepared by those skilled in the art by recombinant techniques. Nucleic acid sequences encoding such fusion proteins can be ordered from any of the various commercial suppliers.The nucleic acid sequence encoding the fusion protein is incorporated into an expression vector functionally linked to one or more expression regulatory elements, the vector is introduced into a suitable host cell, and the fusion protein is isolated from the host cell culture by techniques well known in the art.

[0207] Polymer carrier In some embodiments, the extension of the in vivo duration of action of the inverse monomer and / or its dimer can be achieved by conjugation to one or more polymer support molecules, such as XTEN polymers or water-soluble polymers.

[0208] XTEN Conjugate The inverse monomer and / or dimer may further comprise the XTEN polymer. The XTEN polymer conjugated (chemically or as a fusion protein) to the inverse monomer and / or dimer provides a duration extension similar to PEGylation and can be produced as a recombinant fusion protein in E. coli. XTEN polymers suitable for use in combination with the inverse monomer and / or dimer of this disclosure are provided in Podust, et al. (2016) “Extension of in vivo half-life of biologically active molecules by XTEN protein polymers”, J Controlled Release 240:52-66 and Haeckel et al. (2016) “XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic” PLOS ONE|DOI:10.1371 / journal.pone.0157193 June 13, 2016. The XTEN polymer fusion protein may incorporate a protease-sensitive cleavage site, such as an MMP-2 cleavage site, between the XTEN polypeptide and the inverse monomer and / or dimer.

[0209] Water-soluble polymers In some embodiments, the inverse monomer and / or its dimer may be conjugated into one or more water-soluble polymers. Examples of water-soluble polymers useful for carrying out the present disclosure include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), polyolefin alcohols, poly-alpha-hydroxy acids), polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0210] In some embodiments, the inverse monomer and / or dimer can be conjugated to one or more polyethylene glycol molecules, or "PEGylated." The method or site of PEGylated bond to the binding molecule can vary, but in certain embodiments, PEGylated does not alter, or alters minimally alters, the activity of the binding molecule.

[0211] PEGs suitable for conjugation into polypeptide sequences are generally soluble in water at room temperature and have the following general formula: R(O-CH2-CH2) n Ure The formula has the following characteristics, where R is a hydrogen atom or a protecting group such as an alkyl group or alkanol group, and n is an integer from 1 to 1000. If R is a protecting group, it generally has 1 to 8 carbon atoms. PEG can be linear or branched. Branched PEG derivatives, "star-PEG" and multi-armed PEG are intended by this disclosure.

[0212] In some embodiments, selective PEGylation of inverse monomers and / or dimers may be used, for example, by incorporating non-natural amino acids having side chains to promote selective PEG conjugation. Specific PEGylation sites may be selected so that PEGylation of the binding molecule does not affect its binding to the target receptor.

[0213] In certain embodiments, the increase in half-life is greater than any decrease in biological activity. PEGs suitable for conjugation into polypeptide sequences are generally soluble in water at room temperature and have the general formula R(O-CH2-CH2)nO-R, where R is a hydrogen atom or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. If R is a protecting group, it generally has 1 to 8 carbon atoms. PEG conjugated into polypeptide sequences can be linear or branched. Branched PEG derivatives, "star-PEG," and multi-armed PEGs are intended by this disclosure.

[0214] The molecular weight of PEG used in this disclosure is not limited to any particular range. The PEG component of the binding molecule may have a molecular weight greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. Linear or branched PEG molecules have molecular weights of approximately 2,000 to 80,000 daltons, or approximately 2,000 to 70,000 daltons, or approximately 5,000 to 50,000 daltons, or approximately 10,000 to 50,000 daltons, or approximately 20,000 to 50,000 daltons, or approximately 30,000 to 50,000 daltons, or approximately 20,000 to 40,000 daltons, or approximately 30,000 to 40,000 daltons. In one aspect of this disclosure, the PEG is a 40kD branched PEG containing two 20kD arms.

[0215] In some embodiments, the Disclosure provides “monoPEGylated” inverse monomer dimers (i.e., inverse monomer dimers in which only one of the inverse monomer dimers is PEGylated) and “diPEGylated” inverse monomer dimers (i.e., inverse monomer dimers in which both of the inverse monomer dimers are PEGylated).

[0216] This disclosure also envisions compositions of conjugates in which PEG has various n values ​​and therefore various different PEGs are present in specific ratios. For example, some compositions include mixtures of conjugates where n=1, 2, 3, and 4. In some compositions, the percentage of conjugates with n=1 is 18–25%, the percentage of conjugates with n=2 is 50–66%, the percentage of conjugates with n=3 is 12–16%, and the percentage of conjugates with n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. The conjugate fractions may be decomposed using chromatography, and then, for example, fractions containing conjugates with a desired number of PEGs are identified and purified from the unmodified protein sequence and from the conjugates with other numbers of PEGs.

[0217] PEGs suitable for conjugation into polypeptide sequences are generally soluble in water at room temperature and have the general formula R(O-CH2-CH2) n The formula has OR, where R is a hydrogen atom or a protecting group such as an alkyl group or alkanol group, and n is an integer from 1 to 1000. If R is a protecting group, it generally has 1 to 8 carbon atoms.

[0218] Two widely used first-generation activated monomethoxyPEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) 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. The use of PEG aldehyde linkers targets a single site at the N-terminus of a polypeptide by reductive amination.

[0219] PEGylation most frequently occurs at the N-terminal α-amino group of a polypeptide, the epsilon-amino group of the side chain of a lysine residue, and the imidazole group of the side chain of a histidine residue. Since most recombinant polypeptides have a single α-amino group and numerous epsilon-amino and imidazole groups, a large number of positional isomers can be generated depending on the linker chemistry. General PEGylation strategies known in the art can be applied herein.

[0220] PEG can be attached to the binding molecule of this disclosure via terminal reactive groups ("spacers") that mediate the binding between one or more free amino groups or free carboxyl groups of the polypeptide sequence and polyethylene glycol. PEG having spacers that can bind to free amino groups includes N-hydroxysuccinilimide polyethylene glycol, which can be prepared by activating a succinate ester of polyethylene glycol with N-hydroxysuccinilimide.

[0221] In some embodiments, PEGylation of a binding molecule is facilitated by the incorporation of a non-natural amino acid having a unique side chain that promotes site-specific PEGylation. The incorporation of non-natural amino acids into polypeptides to provide a functional moiety that achieves site-specific PEGylation of such polypeptides is known in the art. For example, see Ptacin et al., PCT International Application No. PCT / US2018 / 045257, filed on 3 August 2018 and published on 7 February 2019 as International Publication No. WO 2019 / 028419A1.

[0222] PEG conjugated to a polypeptide sequence may be linear or branched. Branched PEG derivatives, "star-PEG," and multi-armed PEG are intended by this disclosure.Specific embodiments of PEG useful for implementing this disclosure include 10kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601). (USA), 10kDa linear PEG-NHS esters (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20kDa linear PEG-aldehydes (e.g., Sunbright® ME-200AL, NOF), 20kDa linear PEG-NHS esters (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), 20kDa 2-arm branched PEG-aldehyde containing two 10kDa linear PEG molecules. PEG-aldehyde (e.g., Sunbright® GL2-200AL3, NOF), 20kDa PEG-NHS esters containing two 10kDa linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), 40kDa PEG-aldehyde (e.g., Sunbright® GL2-400AL3), 40kDa PEG-NHS esters containing two 20kDa linear PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30kDa It includes PEG-aldehydes (e.g., Sunbright® ME-300AL) and linear 30kDa PEG-NHS esters.

[0223] In some embodiments, a linker can be used to link the inverse monomer with the PEG molecule. Preferred linkers include “flexible linkers” that are generally long enough to allow some movement between the modified polypeptide sequence and the linking component and molecule. Linker molecules are generally about 6 to 50 atomic lengths. Linker molecules can also be, for example, arylacetylenes, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacitors, amino acids, or combinations thereof. Preferred linkers can be easily selected and may be of any preferred length, e.g., 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 30, 30 to 50, or more than 50 amino acids. Examples of flexible linkers are described in Section IV. Furthermore, multimers of these linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) may be linked together to provide a flexible linker that can be used to conjugate two molecules. Instead of polypeptide linkers, the linker may be a chemical linker, such as a PEG-aldehyde linker. In some embodiments, the binding molecule is acetylated at its N-terminus by an enzymatic reaction with an N-terminal acetyltransferase, and for example, acetyl-CoA. Alternatively, in addition to N-terminal acetylation, the binding molecule may be acetylated at one or more lysine residues by an enzymatic reaction with, for example, a lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325(5942):834-840.

[0224] In some aspects, the Disclosure provides a PEGylated inverse monomer in which PEG is conjugated to an inverse monomer, and PEG is a linear or branched PEG molecule having a molecular weight of about 2,000 to about 80,000 daltons, or about 2,000 to about 70,000 daltons, or about 5,000 to about 50,000 daltons, or about 10,000 to about 50,000 daltons, or about 20,000 to about 50,000 daltons, or about 30,000 to about 40,000 daltons, or about 30,000 to about 40,000 daltons. In one aspect of the Disclosure, PEG is a 40kD branched PEG containing two 20kD arms.

[0225] In some embodiments, the present disclosure provides a PEGylated inverse monomer, the PEGylated inverse monomer being of formula 2 or 3: PEG-L m -[Inverse monomer] [2] [Inverse Monomer]-L m -PEG [3] It is the molecule of, in the formula, (a) PEG is a linear or branched polyethylene glycol molecule having a molecular weight of approximately 10 kD to approximately 80 kD. (v) The inverse monomer is the inverse monomer of equation 1, (c)L is a polypeptide linker or chemical linker, (d) and m = 0 (does not exist) or 1 (exists), In some embodiments, the PEGylated inverse monomer of formula 2 or 3 comprises a 40 kDa linear or branched PEG. In some embodiments, the 40 kDa PEG is a branched 40 kDa PEG containing two 20 kDa arms. In some embodiments, the PEGylated inverse monomer of formula 2 or 3 has the following structure: Includes a 40kDa branched PEG in TIFF2026519565000059.tif17128.

[0226] In some embodiments, the reverse monomer of the present disclosure is a PEGylated reverse monomer of formula 2, wherein (a) the reverse monomer is a polypeptide selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:11, and (b) m=1 and PEG has the following structure: This is a 40kDa branched PEG file from TIFF2026519565000060.tif17128.

[0227] In some embodiments, the reverse monomer of the present disclosure is a PEGylated reverse monomer of formula 3, wherein (a) the reverse monomer is a polypeptide selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:11, and (b) m=1 and PEG has the following structure: This is a 40kDa branched PEG file from TIFF2026519565000061.tif17128.

[0228] Fatty acid carriers In some embodiments, inverse monomers and / or dimers that have an extended duration of action in mammalian subjects and are useful for carrying out the disclosure are realized by covalent bonding of the inverse monomer and / or dimer to a fatty acid molecule, as described in Resh (2016) Progress in Lipid Research 63:120-131. Examples of fatty acids that can be conjugated include myristate, palmitate, and palmitoleic acid. Myristoylates are typically linked to N-terminal glycine, but lysine may also be myristoylated. Palmitoylation is typically achieved by enzymatic modification of a free cysteine-SH group, such as by a DHHC protein that catalyzes S-palmitoylation. Palmitoylation of serine and threonine residues is typically achieved enzymatically using the PORCN enzyme. In some embodiments, the inverse monomer and / or dimer are acetylated at the N-terminus by enzymatic reaction with an N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, the reverse monomer and / or its dimer may be acetylated at one or more lysine residues by enzymatic reaction with lysine acetyltransferase, for example. See, for example, Choudhary et al. (2009) Science 325(5942):834L2 ortho840.

[0229] Recombinant production In some embodiments, the inverse monomers and / or dimers of the present disclosure are produced by recombinant DNA technology. In a typical implementation of recombinant polypeptide production, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector suitable for the host cell in which expression is to be achieved, and the nucleic acid sequence is functionally ligated to one or more expression regulatory sequences encoded by the vector and functional in the target host cell. The recombinant protein can be recovered by disruption of the host cell or, if a secretory leader sequence (signal peptide) is incorporated into the polypeptide, from the cell culture medium.

[0230] In certain embodiments, the inverse monomer and / or dimer of the Disclosure includes an amino acid substitution resulting in enhanced recombinant expression compared to the expression of wild-type hIL10 or mutein without such substitution; a pharmaceutical composition comprising the inverse monomer and / or dimer; a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the inverse monomer and / or dimer; recombinant cells engineered to express the inverse monomer and / or dimer; and a kit comprising the inverse monomer and / or dimer, the nucleic acid encoding the inverse monomer and / or dimer, or recombinant cells expressing the inverse monomer and / or dimer.

[0231] Nucleic acid sequences encoding the inverse monomer and / or its dimer: In some embodiments, the inverse monomer and / or dimer is produced by a recombinant method using a nucleic acid sequence encoding the inverse monomer and / or dimer (or a fusion protein containing the inverse monomer and / or dimer). The nucleic acid sequence encoding the desired inverse monomer and / or dimer can be synthesized by chemical means using an oligonucleotide synthesizer.

[0232] In some embodiments, the inverse monomer and / or dimer is produced by a recombinant method using a nucleic acid sequence encoding the inverse monomer and / or dimer (or a fusion protein containing the inverse monomer and / or dimer). The nucleic acid sequence encoding the desired inverse monomer and / or dimer can be synthesized by chemical means using an oligonucleotide synthesizer.

[0233] Nucleic acid molecules are not limited to sequences that encode polypeptides, but can also include some or all of non-coding sequences upstream or downstream of the coding sequence. Those skilled in molecular biology are familiar with common procedures for isolating nucleic acid molecules. They can be produced, for example, by treating genomic DNA with restriction endonucleases or by performing polymerase chain reactions (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0234] Nucleic acid molecules encoding inverse monomers and / or their dimers (and their fusions) may contain sequences that are naturally occurring or sequences that are different from naturally occurring sequences but which encode the same polypeptide due to the degeneracy of the genetic code. These nucleic acid molecules may consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., those produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, nucleic acid molecules may be double-stranded or single-stranded (i.e., either sense strands or antisense strands).

[0235] Nucleic acid sequences encoding inverse monomers and / or dimers can be obtained from various commercial suppliers that provide custom-made nucleic acid sequences. The amino acid sequence variants of the inverse monomers and / or dimers of this disclosure can be prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code known in the art. Such variants represent insertions, substitutions, and / or specific deletions of the described residues. Any combination of insertions, substitutions, and / or specific deletions is made to arrive at the final construct, insofar as the final construct has the desired biological activity as defined herein.

[0236] Methods for constructing DNA sequences encoding the inverse monomer and / or dimer of the disclosure and expressing those sequences in a suitably transformed host include, but are not limited to, the use of PCR-assisted mutagenesis techniques. Mutations consisting of deletions or additions of amino acid residues to the inverse monomer and / or dimer can also be produced using standard recombination techniques. In the case of deletions or additions, the nucleic acid molecule encoding the inverse monomer and / or dimer is optionally digested with a suitable restriction endonuclease. The resulting fragment can be expressed directly or further manipulated, for example, by ligating it into a second fragment. Ligation may be facilitated if the two ends of the nucleic acid molecule contain complementary nucleotides that overlap each other, but blunt-end fragments can also be ligated. Various mutant sequences can also be generated using PCR-generated nucleic acids.

[0237] The inverse monomers and / or dimers of this disclosure may be synthesized directly through recombinant production, or they may be synthesized as fusion polypeptides with heterologous polypeptides, such as signal sequences, or other polypeptides having specific cleavage sites at the N-terminus or C-terminus of the inverse monomer. In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding a signal peptide. Generally, the signal sequence may be a component of a vector or part of a coding sequence inserted into a vector. The selected heterologous signal sequence is preferably one that is recognized and processed by a host cell (i.e., cleaved by a signal peptidase). The inclusion of the signal sequence depends on whether it is desirable for the recombinant cell from which it is produced to secrete the inverse monomers and / or dimers. If the selected cell is a prokaryotic cell, it is generally preferable that the DNA sequence does not encode a signal sequence. If the recombinant host cell is a yeast cell such as Saccharomyces cerevisiae, an alpha-conjugation factor secretion signal sequence may be used to achieve extracellular secretion of the inverse monomer and / or its dimer into the culture medium, as described in Singh, U.S. Patent No. 7,198,919, issued April 3, 2007. In some embodiments, the signal peptide comprises an endogenous or wild-type IL10 signal peptide. In some embodiments, the signal peptide is the amino acid sequence of a human IL10 polypeptide: Includes TIFF2026519565000062.tif4128. In some embodiments, the signal peptide is the amino acid sequence of the mouse IL10 polypeptide: Includes TIFF2026519565000063.tif4128.

[0238] When an inverse monomer and / or dimer is expressed as a chimera (e.g., a fusion protein comprising an inverse monomer and / or dimer and a heterologous polypeptide sequence), the chimeric protein may be encoded by a hybrid nucleic acid molecule comprising a first sequence encoding all or part of the inverse monomer and / or dimer and a second sequence encoding all or part of the heterologous polypeptide. For example, the inverse monomer and / or dimer of the subject matter described herein may be fused to a hexa- / octahistidine tag (disclosed as SEQ ID NO 139-140, "HHHHHH" and "HHHHHHHH", respectively) to facilitate the purification of bacterial-expressed proteins, or to a hemagglutinin tag to facilitate the purification of proteins expressed in eukaryotic cells. Firstly and secondly, the heterologous polypeptide may be ligated to either the N-terminus and / or C-terminus of the inverse monomer and / or dimer, and should not be understood as being limited to the orientation of the elements of the fusion protein. For example, the N-terminus may be linked to a targeting domain, and the C-terminus may be linked to a hexahistidine (His6) tag (SEQ ID NO: 139) purification handle.

[0239] A reverse-translated gene can be constructed using the complete amino acid sequence of the polypeptide (or fusion / chimer) to be expressed. DNA oligomers containing nucleotide sequences encoding the reverse monomer and / or dimer can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain a 5' or 3' overhang for complementary assembly.

[0240] Codon optimization: In some embodiments, nucleic acid sequences encoding inverse monomers and / or dimers can be “codon-optimized” to enhance expression within specific host cell types. Techniques for codon optimization in a wide variety of expression systems, including mammalian, yeast, and bacterial host cells, are well known, and online tools exist to provide codon-optimized sequences for expression in various host cell types. See, for example, Hawash, et al., (2017) 9:46-53, and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols, edited by David Hacker (Human Press New York). In addition, there are various web-based online software packages freely available to assist in the preparation of codon-optimized nucleic acid sequences.

[0241] Expression vector: After construction (by synthesis, site-directed mutagenesis, or other means), nucleic acid sequences encoding the inverse monomer and / or its dimer can be inserted into an expression vector. Various expression vectors are available for use in different host cells and are typically selected based on the host cell for expression. An expression vector typically contains, but is not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, and integration vectors. Plasmids are an example of non-viral vectors.

[0242] In some embodiments, for example, in relation to a heterodimer inverse monomer, where the first and second inverse monomers of the dimer are different, the vector comprises a first nucleic acid sequence encoding the first inverse monomer and a second nucleic acid sequence encoding the second inverse monomer, the first and second nucleic acid sequences functionally linked to an expression regulatory element (e.g., a promoter), and the first and second nucleic acid sequences are separated by a sequence that promotes co-expression (e.g., an IRES sequence or a T2A sequence). Alternatively, the vector comprises a first nucleic acid sequence encoding the first inverse monomer and a second nucleic acid sequence encoding the second inverse monomer, the first and second nucleic acid sequences functionally linked to expression regulatory sequences, which may be the same or different.

[0243] To promote the efficient expression of recombinant polypeptides, the nucleic acid sequence encoding the polypeptide sequence to be expressed can be functionally ligated to transcriptional and translational regulatory sequences that are functional within a selected expression host.

[0244] Selection marker: Expression vectors typically contain a selection gene, also known as a selection marker. This gene encodes a protein necessary for the survival or proliferation of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for nutritional deficiencies; or (c) supply essential nutrients that are not available from the combined medium.

[0245] Regulatory array: The expression vectors for the inverse monomer and / or dimer of the present disclosure contain regulatory sequences that are recognized by the host organism and functionally linked to the nucleic acid sequence encoding the inverse monomer and / or dimer. The terms “regulatory sequence,” “regulatory sequence,” or “expression regulatory sequence” are used herein without distinction to refer to promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, USA). Regulatory sequences include those that lead to the expression of a nucleotide sequence in many types of host cells and those that induce the expression of a nucleotide sequence only in specific host cells (e.g., tissue-specific regulatory sequences). It will be understood by those skilled in the art that the design of an expression vector may depend on factors such as the selection of the host cell to be transformed and the desired level of protein expression. Various factors understood by those skilled in the art should be considered in the selection of the expression regulatory sequence. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with actual DNA sequences encoding the inverse monomer and / or dimer of the subject, particularly with respect to potential secondary structures.

[0246] promoter: In some embodiments, regulatory sequences are, for example, promoters selected based on the cell type to which expression is desired. Promoters are untranslated sequences located upstream (5') (generally within about 100–1000 bp) of the start codon of structural genes that control the transcription and translation of specific nucleic acid sequences to which they are functionally linked. Such promoters are typically classified into two classes: inductive and constitutive. Inductive promoters are those that initiate an increased level of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or changes in temperature. A large number of promoters recognized by various potential host cells are well known.

[0247] In bacteria, the T7 promoter can be used; in insect cells, the polyhedrin promoter can be used; and in mammalian cells, the cytomegalovirus promoter or metallothionein promoter can be used. Furthermore, in higher eukaryotes, tissue-specific and cell-type-specific promoters are widely available. These promoters are so named for their ability to induce the expression of nucleic acid molecules within a given tissue or cell type in the body. Those skilled in the art are familiar with the numerous promoters and other regulatory elements that can be used to induce nucleic acid expression.

[0248] Transcription from a vector in mammalian host cells may be controlled by promoters obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (e.g., human adenovirus serotype 5), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retrovirus (e.g., mouse stem cell virus), hepatitis B virus, most preferably Simian virus 40 (SV40), heterozoan promoters, such as actin promoters, PGK (phosphoglycerate kinase), or immunoglobulin promoters, or heat shock promoters, if such promoters are compatible with the host cell line. Early and late promoters of the SV40 virus can be conveniently obtained as SV40 restriction fragments that also contain the SV40 virus origin of replication.

[0249] Enhancer: Transcription in higher eukaryotes is often increased by inserting enhancer sequences into vectors. Enhancers are typically cis-acting elements of DNA, usually about 10–300 bp in length, that act on promoters to increase their transcription. Enhancers are relatively independent of orientation and position and can be found at the 5' and 3' ends of the transcription unit, within introns, and within the coding sequence itself. Many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically, enhancers derived from eukaryotic viruses are used. Examples include the SV40 enhancer located late at the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located late at the origin of replication, and the adenovirus enhancer. Enhancers may be spliced ​​and inserted into the expression vector at the 5' or 3' end of the coding sequence, but preferably located at the 5' end from the promoter. Expression vectors used in eukaryotic host cells also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are commonly available from the 5' untranslated region and, optionally, the 3' untranslated region of eukaryotic or viral DNA or cDNA. Standard techniques are used in the construction of suitable vectors containing one or more of the components listed above.

[0250] In addition to sequences that promote the transcription of inserted nucleic acid molecules, vectors may contain origins of replication and other genes encoding selection markers. For example, the neomycin resistance (neoR) gene confers G418 resistance to cells expressing it, thus enabling phenotypic selection of transfected cells. Examples of additional marker or reporter genes include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art can easily determine whether a given regulatory element or selection marker is suitable for use in a particular experimental situation.

[0251] The proper construction of an expression vector can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host.

[0252] Host cells: The disclosure further provides prokaryotic or eukaryotic cells containing and expressing one or more nucleic acid molecules encoding an inverse monomer and / or its dimer. The cells of the disclosure are transfected cells, i.e., cells into which nucleic acid molecules, such as nucleic acid molecules encoding an inverse monomer and / or its dimer, have been introduced by recombinant DNA technology.

[0253] In some embodiments, for example, in relation to a heterodimer reciprocal monomer, where the first and second reciprocal monomers of the dimer are different, the recombinant modified cell includes a vector comprising a first nucleic acid sequence encoding the first reciprocal monomer and a second nucleic acid sequence encoding the second reciprocal monomer, the first and second nucleic acid sequences functionally linked to a single expression regulatory sequence, and the first and second nucleic acid sequences separated by a sequence that promotes co-expression. In other embodiments, the recombinant modified cell includes a vector comprising a first nucleic acid sequence encoding the first reciprocal monomer and a second nucleic acid sequence encoding the second reciprocal monomer, the first and second nucleic acid sequences functionally linked to expression regulatory sequences, respectively. In other embodiments, for example, in relation to heterodimeric inverse monomers, if the first and second inverse monomers of the dimer are different, the recombinant modified cell may contain two vectors: the first vector containing a first nucleic acid sequence encoding the first inverse monomer, functionally linked to an expression regulatory sequence; and the second vector containing a nucleic acid sequence encoding the second inverse monomer. In some embodiments, the recombinant modified cell is a prokaryotic cell, such as a bacterial cell. In some embodiments, the recombinant modified cell is a eukaryotic cell, such as a mammalian cell.

[0254] Host cells are typically selected according to their compatibility with the selected expression vector, the toxicity of the products encoded by the DNA sequence of the present invention, their secretory properties, their ability to properly fold polypeptides, their fermentation or culture requirements, and the ease of purifying the products encoded by the DNA sequence. Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotic cells, yeast cells, or higher eukaryotic cells.

[0255] In some embodiments, recombinant inverse monomers can also be produced within eukaryotes such as yeast cells or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors usable for protein expression in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Examples include pPicZ (Invitrogen Corporation, San Diego, Calif.) and pPicZ (Invitrogen Corporation, San Diego, Calif.); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.6:187:195)).

[0256] Examples of useful mammalian host cell lines include mouse L cells (LM[TK-], ATCC#CRL-2648), monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human fetal kidney cell line (HEK293 cells, or HEK293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep Examples include G2, HB 8065); mouse mammary gland tumors (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2). In mammalian cells, the regulatory function of expression vectors is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and Simianvirus 40.

[0257] Inverse monomers and / or dimers can be produced in prokaryotic hosts, e.g., *Escherichia coli*, or in eukaryotic hosts, e.g., insect cells (e.g., Sf21 cells) or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many suppliers, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, only the compatibility of the components with each other is important. Those skilled in the art can make such a decision. Furthermore, if guidance is needed in selecting an expression system, those skilled in the art can refer to Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0258] In some embodiments, the resulting inverse monomer is glycosylated or deglycosylated depending on the host organism used to produce the polypeptide. When bacteria are selected as the host, the produced inverse monomer is deglycosylated. Eukaryotic cells, on the other hand, typically result in glycosylation of the inverse monomer.

[0259] In some embodiments, the amino acid sequence (particularly the CDR sequence) of sdAb incorporated into the inverse monomer and / or its dimer may contain a glycosylation motif, particularly an N-linked glycosylation motif of the sequence Asn-X-Ser(NXS) or Asn-X-Thr(NXT), where X is any amino acid other than proline. In such cases, it is desirable to eliminate such an N-linked glycosylation motif by modifying its sequence to prevent glycosylation. In some embodiments, the N-linked glycosylation motif is disrupted by incorporating a conserved amino acid substitution of the Asn(N) residue of the N-linked glycosylation motif.

[0260] For other additional expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY). See also Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0261] Transfection: Expression constructs can be introduced into host cells to produce the inverse monomer and / or dimer disclosed herein. Expression vectors containing nucleic acid sequences encoding the inverse monomer and / or dimer can be introduced into prokaryotic or eukaryotic host cells by conventional transformation or transfection techniques. Preferred methods for transforming or transfecting host cells can be found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals. To facilitate transfection of target cells, target cells may be directly exposed to a nonviral vector under conditions that promote the uptake of the nonviral vector. Examples of conditions that promote the uptake of foreign nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (Lipofectamine®, Thermo-Fisher Scientific, etc.), high salt levels, and magnetic fields (electroporation).

[0262] Cell culture: Cells can be cultured in conventional nutrient media appropriately modified for promoter induction, transformant selection, or amplification of genes encoding desired sequences. Mammalian host cells can be cultured in a variety of media. Commercial media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or equivalent energy sources. Any other necessary adjuvants may also be included in appropriate concentrations that would be known to those skilled in the art. Culture conditions such as temperature and pH, for example, have been previously used with host cells selected for expression and would be obvious to those skilled in the art.

[0263] Recombinant protein recovery: When a secreted leader sequence is used, the recombinantly produced inverse monomer and / or dimer can be recovered from the culture medium as secreted polypeptides. Alternatively, the inverse monomer and / or dimer can be recovered from host cell lysates. To inhibit proteolysis during purification, protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) may be used during the recovery period from cell lysates, and antibiotics may be included to prevent the growth of exogenous contaminants.

[0264] Various purification processes, such as affinity chromatography, are known and used in the art. Affinity chromatography utilizes highly specific binding sites typically present in biological macromolecules and separates molecules using their ability to bind to specific ligands. Covalent bonding explicitly presents the ligand to the protein sample, thereby attaching the ligand to an insoluble porous support medium in a manner that separates and purifies a second species from a mixture using the innate specific binding of one molecular species. Antibodies are commonly used in affinity chromatography. Size selection processes may also be used; for example, gel filtration chromatography (also known as size exclusion chromatography or molecular sieve chromatography) is used to separate proteins according to their size. In gel filtration, a protein solution is passed through a column packed with a semipermeable porous resin. The semipermeable resin has a range of pore sizes that determine the size of the proteins that can be separated using the column.

[0265] Recombinant inverse monomers and / or dimers expressed by a transformed host can be purified according to any preferred method. Recombinant inverse monomers and / or dimers can be isolated from inclusion bodies produced in Escherichia coli or from conditioned media derived from mammalian or yeast cultures producing a given polypeptide, using cation exchange, gel filtration, and / or reversed-phase liquid chromatography. Substantially purified forms of recombinant inverse monomers and / or dimers can be purified from the expression system using common biochemical procedures and used, for example, as therapeutic agents as described herein.

[0266] In some embodiments, when the inverse monomer and / or dimer is expressed with the purification tag described above, this purification handle can be used to isolate the inverse monomer and / or dimer from cell lysates or cell culture media. If the purification tag is a chelate peptide, methods for isolating such molecules using immobilized metal affinity chromatography are well known in the art. See, for example, Smith, et al., U.S. Patent No. 4,569,794.

[0267] The biological activity of the recovered inverse monomer and / or dimer can be assayed for activity by any suitable method known in the art, and can be evaluated in substantially purified form or as part of a cell lysate or cell culture medium when a secretory leader sequence is used for expression.

[0268] Pharmaceutical preparations In some embodiments, the inverse monomer and / or dimer of a subject (and / or nucleic acids encoding the inverse monomer and / or dimer, or recombinant cells modified to incorporate nucleic acid sequences and express the inverse monomer and / or dimer) may be incorporated into a composition comprising a pharmaceutical composition. Such compositions typically comprise a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. The pharmaceutical composition is formulated to be compatible with its intended route of administration and is suitable for therapeutic use in which the inverse monomer and / or dimer will be administered to a subject in need of treatment.

[0269] Carrier: The carrier may include sterile diluents, such as sterile water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant, such as sodium dodecyl sulfate. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS).

[0270] Buffer: The term buffer solution includes buffers such as acetates, citrates, or phosphates, and active ingredients for adjusting the tonicity, such as sodium chloride or dextrose. pH can be adjusted using acids or bases such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide (for example, to about 7.2–7.8, e.g., up to pH 7.5).

[0271] Dispersion: Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for preparing sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield the active ingredient in powder form and any additional desired components from its pre-sterilized filtered solution.

[0272] Preservatives: Pharmaceutical formulations for parenteral administration to subjects should be sterile and fluid to facilitate syringability. They should be stable under manufacturing and storage conditions and protected against contamination. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as active agents like benzyl alcohol or methylparaben; antioxidants like ascorbic acid or sodium bisulfite; and chelating agents like ethylenediaminetetraacetic acid, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Sterile solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent along with one or a combination of the components listed above, as needed, followed by filtration sterilization.

[0273] Tonicity agent: In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, in the composition.

[0274] Route of administration: Some aspects of the therapeutic methods of the present disclosure involve the administration of a pharmaceutical formulation containing an inverse monomer and / or its dimer (and / or a nucleic acid encoding the inverse monomer and / or its dimer, or recombinant modified host cells expressing the inverse monomer and / or its dimer) to a subject in need of treatment. The pharmaceutical formulations containing the inverse monomer and / or its dimer of the present disclosure may be administered to a subject in need of treatment or prevention by a variety of routes of administration, including parenteral administration, oral routes, topical routes, or inhalation routes.

[0275] Parenteral administration: In some embodiments, the methods of the present disclosure involve parenteral administration of a pharmaceutical formulation comprising an inverse monomer and / or its dimer (and / or nucleic acids encoding the inverse monomer and / or its dimer, or recombinant modified host cells expressing the inverse monomer and / or its dimer) to a subject in need of treatment. Examples of parenteral administration routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. The parenteral formulation comprises a solution or suspension used for parenteral administration and may include a vehicle, carrier, and buffer. Pharmaceutical formulations for parenteral administration include a sterile aqueous solution (if water-soluble) or dispersion, and a sterile powder for immediate preparation of a sterile injection solution or dispersion. Parenteral preparations may be sealed in ampoules, disposable syringes, or glass or plastic multi-dose vials. In one embodiment, the formulation is provided in a pre-filled syringe.

[0276] Oral administration: In some embodiments, the methods of the present disclosure involve the oral administration of a pharmaceutical formulation comprising an inverse monomer and / or its dimer (and / or nucleic acids encoding the inverse monomer and / or its dimer, or recombinant modified host cells expressing the inverse monomer and / or its dimer) to a subject in need of treatment. The oral composition, if used, generally includes an inert diluent or food carrier. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. The oral composition may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties, namely, binders, e.g., microcrystalline cellulose, tragacanth gum, or gelatin; excipients, e.g., starch, or lactose; disintegrants, e.g., alginic acid, Primogel®, or corn starch; lubricants, e.g., magnesium stearate, or Sterotes®; lubricants, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose, or saccharin; or flavoring agents, e.g., peppermint, methyl salicylate, or orange flavoring.

[0277] Inhalation preparations: In some embodiments, the methods of the present disclosure involve the inhalation administration of a pharmaceutical formulation comprising an inverse monomer and / or its dimer (and / or a nucleic acid encoding the inverse monomer and / or its dimer, or recombinant modified host cells expressing the inverse monomer and / or its dimer) to a subject in need of treatment. In the case of inhalation administration, the inverse monomer and / or its dimer of the subject, or the nucleic acid encoding it, is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a gas such as carbon dioxide, or from a nebulizer. Such methods include the method described in U.S. Patent No. 6,468,798.

[0278] Mucosal preparations and transdermal preparations: In some embodiments, the methods of the present disclosure involve mucosal or transdermal administration of a pharmaceutical formulation containing an inverse monomer and / or its dimer (and / or nucleic acids encoding the inverse monomer and / or its dimer, or recombinant modified host cells expressing the inverse monomer and / or its dimer) to a subject in need of treatment. In the case of transmucosal or transdermal administration, a penetration agent suitable for the barrier to be penetrated is used in the formulation. Such penetration agents are generally known in the art and, for example, in the case of transmucosal administration, include detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be achieved by the use of nasal sprays or suppositories (including conventional suppository bases such as cocoa butter and other glycerides, for example), or retained enemas for rectal delivery. In the case of transdermal administration, the active compound is formulated in the form of an ointment, plaster, gel or cream, as is generally known in the art, and may incorporate a penetration enhancer such as ethanol or lanolin.

[0279] Sustained-release formulations and depot formulations: In some aspects of the methods of this disclosure, the inverse monomer and / or its dimer are administered in formulation to a subject in need of treatment to provide sustained release of the inverse monomer and / or its dimer. Examples of sustained-release formulations of injectable compositions may be brought about by including in the composition an active agent that delays absorption, such as aluminum monostearate and gelatin. In one aspect, the inverse monomer and / or its dimer or nucleic acid of the subject is prepared using a controlled-release formulation comprising a carrier, such as an implant and a microencapsulation delivery system, to protect the inverse monomer and / or its dimer from rapid elimination from the body. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Such formulations can be prepared using standard techniques. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes that target infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared, for example, as described in U.S. Patent No. 4,522,811, according to methods known to those skilled in the art.

[0280] Administration of nucleic acids encoding inverse monomers and / or dimers In some aspects of the methods of this disclosure, delivery of the inverse monomer and / or its dimer to a subject in need of treatment is achieved by administration of nucleic acids encoding the inverse monomer and / or its dimer. Methods of administering nucleic acids encoding the inverse monomer and / or its dimer to a subject include, but are not limited to, transfection or infection using methods known in the art, including methods described in McCaffrey et al. (Nature (2002) 418:6893), Xia et al. (Nature Biotechnol. (2002) 20:1006-1010) or Putnam (Am.J. Health Syst. Pharm. (1996) 53:151-160 erratum at Am.J. Health Syst. Pharm. (1996) 53:325). In some embodiments, the inverse monomer and / or dimer are administered to a subject by administration of a pharmaceutically acceptable formulation of a recombinant expression vector, which comprises a nucleic acid sequence encoding the inverse monomer and / or dimer, functionally linked to one or more functional regulatory sequences in a mammalian subject. In some embodiments, functional regulatory sequences within a limited range of cell types (or single cell types) may be selected to promote selective expression of the inverse monomer and / or dimer within a particular target cell type. In one embodiment, the recombinant expression vector is a viral vector. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a replication-deficient adenovirus derived from recombinant adeno-associated virus (rAAV) or recombinant adenovirus (rAd), particularly human adenovirus serotype 3 and / or 5. In some embodiments, the replication-deficient adenovirus has one or more modifications to the E1 region that interfere with the virus's ability to initiate the cell cycle and / or apoptotic pathway in human cells. A replication-deficient adenovirus vector may optionally contain a deletion in the E3 domain. In some embodiments, the adenovirus is a replication-competent adenovirus.In some embodiments, adenoviruses are recombinant viruses with replication ability that have been engineered to selectively replicate within target cell types.

[0281] In some embodiments, particularly for the treatment of intestinal diseases or bacterial infections of a target, nucleic acids encoding the inverse monomer and / or dimer may be delivered to the target by administration of recombinant modified bacteriophage vectors encoding the inverse monomer and / or dimer. Where used herein, the terms “prokaryotic virus,” “bacteriophage,” and “phage” are used without distinction to represent any of the various bacterial viruses that infect and replicate within bacteria. Bacteriophages selectively infect prokaryotic cells and restrict the expression of the inverse monomer and / or dimer in prokaryotic cells in the target, while avoiding expression in mammalian cells. A wide variety of bacteriophages capable of selecting a broad range of bacterial cells have been widely identified and characterized in the scientific literature. In some embodiments, phages are modified to remove adjacent motifs (PAMs). Removing the Cas9 sequence from the phage genome reduces the ability of the target prokaryotic cell's Cas9 endonuclease to neutralize the invading phage encoding the inverse monomer and / or dimer.

[0282] Administration of recombinant modified cells expressing the inverse monomer and / or dimer. In some aspects of the methods of this disclosure, delivery of the inverse monomer and / or its dimer to a subject requiring treatment is achieved by administration of recombinant host cells modified to express the inverse monomer and / or its dimer. Recombinant host cells may be administered in therapeutic and prophylactic applications as described herein. In some aspects, the recombinant host cells are mammalian cells, e.g., human cells. In some aspects, the recombinant host cells are prokaryotic cells, e.g., bacterial cells associated with the gut flora, such as Escherichia coli or Lactobacillus lactis.

[0283] In some embodiments, nucleic acid sequences encoding the inverse monomer and / or its dimer (or vectors containing the same) may be maintained extrachromosomally in recombinantly modified host cells for administration. In other embodiments, nucleic acid sequences encoding the inverse monomer and / or its dimer may be incorporated into the genome of the administered host cell using at least one endonuclease to facilitate the incorporation and insertion of the nucleic acid sequence into the cell's genomic sequence. As used herein, the term “endonuclease” is used to refer to wild-type or variant enzymes that can catalyze the cleavage of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule. An endonuclease is called a “rare-cutting” endonuclease if such an endonuclease has a polynucleotide recognition site that is more than about 12 base pairs (bp), more preferably 14–55 bp in length. Rare-cutting endonucleases can be used to inactivate genes at a locus, or to incorporate a transgene by homologous recombination (HR), that is, by inducing a DNA double-strand break (DSB) at the locus and inserting exogenous DNA into this locus by gene repair mechanisms. Examples of rare-cutting endonucleases include homing endonucleases (Grizot, et al (2009) Nucleic Acids Research 37(16):5405-5419), chimeric zinc finger nucleases (ZFNs) arising from the fusion of manipulated zinc finger domains (Porteus M and Carroll D., Gene targeting using zinc finger nucleases (2005) Nature Biotechnology 23(3):967-973), TALEN nucleases, Cas9 endonucleases derived from the CRISPR system, or modified restriction endonucleases for extended sequence specificity (Eisenschmidt, et al. 2005;33(22):7039-7047).

[0284] In some embodiments, particularly for the administration of the inverse monomer and / or its dimer to the intestinal tract, the inverse monomer and / or its dimer may be delivered to the target by recombinantly modified prokaryotic cells (e.g., Lactobacillus lactis). The use of engineered prokaryotic cells for the delivery of recombinant proteins to the intestinal tract is known in the art. See, for example, Lin, et al. (2017) Microb Cell Fact 16:148. In some embodiments, engineered bacterial cells expressing the inverse monomer and / or its dimer may be administered orally, typically in an aqueous suspension, or rectally (e.g., by enema).

[0285] How to use This disclosure further provides a method for treating subjects suffering from a disease, disorder, or condition by administering a therapeutically effective dose of a recombinant prokaryotic cell or recombinant mammalian cell containing (a) a reverse IL10 monomer and / or its dimer, (b) a pharmaceutically acceptable formulation containing the reverse IL10 monomer and / or its dimer as an active ingredient, (c) a recombinant nonviral vector, a recombinant eukaryotic vector, or a recombinant bacteriophage vector comprising a nucleic acid sequence encoding the reverse monomer and / or its dimer functionally linked to one or more regulatory sequences, (d) a recombinant prokaryotic cell or recombinant mammalian cell genomically modified to contain a nucleic acid sequence encoding the reverse monomer and / or its dimer functionally linked to one or more regulatory sequences, or (e) a recombinant prokaryotic cell or recombinant mammalian cell comprising a nucleic acid encoding the reverse monomer and / or its dimer functionally linked to one or more regulatory sequences.

[0286] Administration: The biased IL10 reverse monomer (and its dimer) of this disclosure exhibits a broader therapeutic range compared to wild-type IL10. “Therapeutic range” refers to a dosage range of the biased IL10 reverse monomer (and / or its dimer) that provides a target concentration of the biased IL10 reverse monomer (or its dimer) sufficient to deliver substantial levels of IL10 activity (e.g., STAT3 signaling) to monocytes, but less than a dose that delivers a target concentration sufficient to induce substantial levels of IL10 activity signaling in T cells. Consequently, in some embodiments, the biased IL10 reverse monomer is administered to a target at a level that delivers a target concentration sufficient to deliver substantial levels of STAT3 signaling to monocytes, but less than a dose that delivers a target concentration sufficient to induce substantial levels of STAT3 signaling in T cells. In some embodiments, the disclosure provides a method for treating a subject suffering from an inflammatory or autoimmune disease, disorder, or condition, characterized by administering a therapeutically effective dose of reverse IL10 monomers and / or dimers thereof (or a pharmaceutically acceptable formulation containing reverse IL10 monomers and / or dimers as active ingredients), wherein the therapeutically effective dose brings to the subject a concentration sufficient to provide substantial levels of STAT3 signaling to monocytes, but less than a dose that brings to the subject a concentration sufficient to induce substantial levels of STAT3 signaling to T cells.

[0287] In one embodiment, the Disclosure provides a method for treating a subject suffering from an inflammatory or autoimmune disease, disorder, or condition by administering a therapeutically effective dose of reverse IL10 monomers and / or dimers (or a pharmaceutically acceptable formulation containing reverse IL10 monomers and / or dimers as active ingredients) at a monomeric dose level that maximizes the ratio of the Emax of IL10 activity on monocytes to the Emax of such IL10 activity on T cells. In several embodiments, the Disclosure provides a method for treating a subject suffering from a disease, disorder, or condition by administering a therapeutically effective dose of reverse IL10 monomers and / or dimers (or a pharmaceutically acceptable formulation containing reverse IL10 monomers and / or dimers as active ingredients), wherein the therapeutically effective dose is (a) STAT3 EC in activated human monocytes 20 In hyper- or activated human monocytes, STAT3 EC 30 In hyper- or activated human monocytes, STAT3 EC 40 In hyper- or activated human monocytes, STAT3 EC 50 In hyper- or activated human monocytes, STAT3 EC 60 In hyper-activated human monocytes, STAT3 EC 70 Alternatively, in activated human monocytes, STAT3 EC 80 In hyper- or activated human monocytes, STAT3 EC 90 In hyper- or activated human monocytes, Emax(EC) 100 (b) is greater than (b) STAT3 EC in activated human T cells 40 In human T cells less than STAT3 EC, or activated human T cells 30 In human T cells less than STAT3 EC, or activated human T cells 20 In human T cells less than STAT3 EC, or activated human T cells 10less than, or a dosage to provide a concentration that is less than STAT3 EC5 in activated human T cells. In some embodiments, the present disclosure provides a method of treating a subject afflicted with a disease, disorder or condition by administering a therapeutically effective amount of reverse IL10 monomer and / or its dimer (or a pharmaceutically acceptable formulation containing reverse IL10 monomer and / or its dimer as an active ingredient), wherein the therapeutically effective amount is greater than STAT3 EC 20 in activated human monocytes but less than STAT3 EC 30 in activated human T cells, or greater than STAT3 EC 50 in activated human monocytes but less than STAT3 EC 20 in activated human T cells, or greater than STAT3 EC 70 in activated human monocytes but less than STAT3 EC 20 in activated human T cells, or greater than STAT3 EC 80 in activated human monocytes but less than STAT3 EC 20 in activated human T cells, or greater than STAT3 Emax in activated human monocytes but less than STAT3 EC 30 in activated human T cells, or greater than STAT3 EC 50 in activated human monocytes but less than STAT3 EC 10 in activated human T cells, or a dosage to provide a concentration that is greater than STAT3 EC in activated human monocytes but less than STAT3 EC in activated human T cells.

[0288] Inflammatory disorders and autoimmune disorders Conditions suitable for treatment with the inverse monomer and / or dimer of the present disclosure (including pharmaceutically acceptable formulations containing the inverse monomer and / or dimer, and / or encoding nucleic acid molecules, including recombinant viruses encoding such inverse monomers and / or dimers) include, but are not limited to, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases including Alzheimer's disease, and systemic lupus erythematosus. Systemic leukemia (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes including type 1 or type 2 diabetes, inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (Seronegativity syndrome) This includes inflammatory or autoimmune diseases such as Enthesopathy (Arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarthritis, polyarthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteritis-associated arthritis, reactive arthritis, Reiter's syndrome, and SEA syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome).

[0289] Other examples of proliferative and / or differentiation disorders suitable for treatment with the inverse monomers and / or dimers of this disclosure (including pharmaceutically acceptable formulations containing the inverse monomers and / or dimers, and / or encoding nucleic acid molecules, including recombinant viruses encoding such inverse monomers and / or dimers) include, but are not limited to, skin disorders. Skin disorders may involve abnormal activity of cells or groups of cells or layers in the dermis, epidermis, or subcutaneous tissue layers, or abnormalities at the dermal-epidermal junction. For example, skin disorders may involve abnormal activity of keratinocytes (e.g., hyperproliferative basal keratinocytes and keratinocytes just above the basal layer), melanocytes, Langerhans cells, Merkel cells, immune cells, and other cells found in one or more of the epidermal layers, e.g., the basal cell layer (germinal layer), spinous layer, granular layer, stratum lucidum, or stratum corneum. In other embodiments, the disorder may be accompanied by abnormal activity of dermal cells in the dermis, such as dermal endothelial cells, fibroblasts, and immune cells (e.g., mast cells or macrophages), such as those found in the dermis, e.g., the papillary or reticular layer.

[0290] Examples of inflammatory or autoimmune skin disorders include psoriasis, psoriatic arthritis, dermatitis (eczema), such as exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosacea, parapsoriasis, lichenoid pityriasis, lichen planus, lichen styloides, and ichthyosiform dermatosis. Skin disorders include dermatosis, keratosis, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, bullous pemphigoid (e.g., ocular scarring pemphigoid or bullous pemphigoid), urticaria, porokeratosis, rheumatoid arthritis with hyperproliferation and inflammation of epithelial-associated cells lining the joint capsule; dermatitis, e.g., seborrheic dermatitis and photodermatitis; keratosis, e.g., seborrheic keratosis, senile keratosis, actinic keratosis, photo-induced keratosis and follicular keratosis; acne vulgaris; keloids and prevention of keloid formation; nevi; warts, condyloma or genital warts, and warts including human papillomavirus (HPV) infections such as sexually transmitted warts; leukoplakia; lichen planus; and keratitis. Skin disorders may include dermatitis, e.g., atopic dermatitis or allergic dermatitis or psoriasis.

[0291] The compositions of this disclosure (including pharmaceutically acceptable formulations comprising inverse monomers and / or dimers thereof, and / or encoding nucleic acid molecules, including recombinant viruses encoding such inverse monomers and / or dimers) may also be administered to patients who have (or may have) psoriasis or psoriatic disorders. The term “psoriasis” is intended to have its medical meaning, namely, a disease that primarily affects the skin, producing raised, thickened, desquamating, and non-scarring lesions. The lesions are typically well-defined erythematous papules covered with shiny, overlapping scales. The scales are typically silvery or slightly milky white. The nails are often affected, resulting in pitting, detachment, thickening, and discoloration. Psoriasis can be associated with arthritis, which can lead to limb impairment. Keratinocyte hyperplasia, along with epidermal inflammation and decreased keratinocyte differentiation, is a key feature of psoriatic epidermal hyperplasia. Several mechanisms have been proposed to explain the keratinocyte hyperproliferation that characterizes psoriasis. Disorders of cellular immunity are also involved in the pathogenesis of psoriasis. Examples of psoriatic disorders include chronic constant psoriasis, plaque psoriasis, moderate to severe plaque psoriasis, plaque psoriasis vulgaris, exanthematous psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or focal pustular psoriasis.

[0292] ulcerative colitis In some embodiments, the Disclosure provides a method for treating a mammalian subject suffering from ulcerative colitis, comprising the step of administering the hIL10 reverse monomer or hIL10 reverse monomer dimer (or a pharmaceutical formulation containing the hIL10 reverse monomer or hIL10 reverse monomer dimer) to a subject, wherein the administration step provides improvement of one or more symptoms of ulcerative colitis. In some embodiments, the method comprises the step of administering the reverse monomer of Formula 1 or Formula 2, or a pharmaceutical formulation containing the reverse monomer of Formula 1 or Formula 2, wherein the administration step provides improvement of one or more symptoms of ulcerative colitis. In one embodiment, the Disclosure provides SEQ ID For polypeptides selected from the group consisting of NO:10, 11, 27, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 53, 108, 109, 110, 111, 112, 113, 114, 46, 47, 48, 49, 50, 51, 52, 53, 54, 121, 122, 123, 124, 125 and 126, at least 90%, or at least 91%, or at least 92%, or at least 93%, The present invention provides a method for treating a mammalian subject suffering from ulcerative colitis, comprising the step of administering an hIL10 reverse monomer or hIL10 reverse monomer dimer having at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity, wherein the administration step provides improvement of one or more symptoms of ulcerative colitis.

[0293] IFNγ-induced anemia: In some embodiments, the Disclosure provides a method for treating and / or preventing IFNγ-induced anemia in a subject, the method comprising the step of administering a therapeutically effective dose of the fused IL10 dimer of the Disclosure to a subject, the administration step resulting in improvement of one or more symptoms of IFNγ-induced anemia in the subject and / or a decrease in serum IFNγ levels. In some embodiments, the Method comprises the step of administering a therapeutically effective dose of the inverse monomer of Formula 1 to a subject, the administration step resulting in improvement of one or more symptoms of IFNγ-induced anemia in the subject and / or a decrease in serum IFNγ levels. IFNγ-induced anemia is a frequent complication in patients with chronic inflammatory diseases. Chronic inflammation can arise from a variety of conditions, such as infectious diseases, AIDS, malignant tumors and / or autoimmune diseases, e.g., inflammatory bowel disease, rheumatoid arthritis. Activated T cells secrete IFNγ and interleukin-2 (IL-2). IFNγ has been observed to increase the cytotoxic effects of TNFα. The compositions of the present disclosure were evaluated in a mouse model of IFNγ-induced anemia (Example 32). In one embodiment, the present disclosure provides a polypeptide selected from the group consisting of SEQ ID NO: 10, 11, 27, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 53, 108, 109, 110, 111, 112, 113, 114, 46, 47, 48, 49, 50, 51, 52, 53, 54, 121, 122, 123, 124, 125 and 126 in a concentration of at least 90%, or at least 91%, or at least 92%, or at least 93%, or The present invention provides a method for treating and / or preventing IFNγ-induced anemia, comprising the step of administering an hIL10 reverse monomer or hIL10 reverse monomer dimer having at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity, wherein the administration step provides improvement of one or more symptoms of IFNγ-induced anemia.

[0294] Macrophage activation syndrome In some embodiments, the Disclosure provides a method for treating and / or preventing macrophage activation syndrome (MAS) of a subject, the method comprising the step of administering a therapeutically effective dose of the reverse IL10 monomer of Formula 1 or Formula 2 of the Disclosure to a subject. Macrophage activation syndrome (MAS) is a potentially life-threatening complication of systemic inflammatory disorders, including, but not limited to, systemic juvenile idiopathic arthritis (sJIA), Kawasaki disease, systemic lupus erythematosus (SLE) and infections, particularly Epstein-Barr virus (EBV) infection, malignancies and primary immunodeficiency disorders. Elevated CD163 is associated with MAS. In some embodiments, the Disclosure provides a method for treating and / or preventing macrophage activation syndrome (MAS) of a subject, the method comprising the step of administering a therapeutically effective dose of the fused IL10 dimer of the Disclosure to a subject, the administration step resulting in improvement of one or more symptoms of macrophage activation syndrome. In one embodiment, the Disclosure provides SEQ ID For polypeptides selected from the group consisting of NO:10, 11, 27, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 53, 108, 109, 110, 111, 112, 113, 114, 46, 47, 48, 49, 50, 51, 52, 53, 54, 121, 122, 123, 124, 125 and 126, at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least The present invention provides a method for treating and / or preventing a target macrophage activation syndrome, comprising the step of administering an hIL10 reverse monomer or hIL10 reverse monomer dimer having 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity, wherein the administration step provides improvement of one or more symptoms of the macrophage activation syndrome.

[0295] As previously mentioned, chronic inflammation is associated with the development of a wide variety of cancers. See, for example, Coussens and Werb (2002) Nature 420:860-867. Consequently, the inverse composition of the present disclosure may be used for the treatment and / or prevention of cancers arising from chronic inflammation. In one embodiment, the cancers associated with chronic inflammation are selected from the group consisting of colorectal cancer, colorectal cancer, pancreatic cancer, and hepatic cancer. In one embodiment, the present disclosure provides a method for preventing the development of cancers associated with chronic inflammation in a human subject by administering a prophylactic effective dose of a composition comprising a vector encoding the inverse hIL10 monomer (or dimer thereof) of the present disclosure, or recombinant cells expressing the inverse hIL10 monomer (or dimer thereof) of the present disclosure, to the subject.

[0296] Methods for modulating IL10-mediated signaling In another aspect, the disclosure provides a method for modulating IL10-mediated signaling in a subject. In some embodiments, the method comprises the step of administering an effective amount of a pharmaceutical composition to a subject, the pharmaceutical composition comprising a recombinant modified cell containing the inverse monomer and / or dimer described herein, a nucleic acid molecule encoding the inverse monomer and / or dimer described herein, or a nucleic acid molecule encoding the inverse monomer and / or dimer described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0297] In some embodiments, a method for modulating IL10-mediated signaling in a subject includes determining STAT3-mediated signaling in one or more cells obtained from the subject. In some embodiments, STAT3-mediated signaling is determined by an assay selected from the group consisting of gene expression assays, phospho-flow signaling assays, and enzyme-linked immunosorbent assays (ELISA). In some embodiments, STAT3-mediated signaling in the subject is reduced by approximately 20% to approximately 100% compared to a reference level. In some embodiments, the administered composition results in a reduction in the ability of the subject to induce the expression of pro-inflammatory genes selected from IFN-γ, granzyme B, granzyme A, perforin, TNF-α, GM-CSF, and MIP1α.

[0298] kit Kits comprising the inverse monomer and / or dimer of the disclosure are also provided. In some embodiments, the kit comprises one or more components for modulating IL10-mediated signaling in a subject or for treating a health condition in a subject requiring such modulation, the components being selected from the inverse monomer and / or dimer described herein, nucleic acid molecules encoding the inverse monomer and / or dimer described herein, recombinant modified cells comprising nucleic acid molecules encoding the inverse monomer and / or dimer described herein, or a pharmaceutical composition comprising one or more components. In some embodiments, the pharmaceutical composition of the kit comprises a pharmaceutically acceptable carrier. [Examples]

[0299] The following examples are provided to illustrate the claimed invention, but are not intended to limit the claimed invention.

[0300] Example 1: Method This example describes a method for preparing the monomers and dimers invented in this disclosure, as well as a method for testing the biophysical properties of the monomers and dimers invented in this disclosure.

[0301] Protein expression and purification Regarding human molecules, natural signal peptides (DNA sequence: TIFF2026519565000064.tif11143; Amino acid sequence: TIFF2026519565000065.tif4128), or mouse Ig heavy chain signal peptide (DNA sequence: TIFF2026519565000066.tif11150; Amino acid sequence: A protein-coding DNA sequence was inserted into pEXSyn 2.0 between the EcoRI restriction site and the NotI restriction site as a C-terminal His tag fusion with one of the following (TIFF2026519565000067.tif4128). The nucleic acid and amino acid sequences of the human and mouse inverse monomers and dimers are shown in Tables 5 to 12.

[0302] Protein expression was induced in expi293 cells by transient transfection. The polypeptide containing the C-terminal His tag was purified from the cell culture supernatant collected using Ni affinity chromatography. The culture supernatant was supplemented with 5 mM imidazole and purified using a gravity column containing Ni-Excel (Cytiva) resin, and equilibrated in PBS containing 5 mM imidazole. Washing and elution were performed in PBS supplemented with 5 mM and 250 mM imidazole, respectively. The proteins were dialyzed and transferred to PBS before storage and testing. Protein purity and quality were evaluated using SD-PAGE, LC-MS, and analytical SEC.

[0303] Protein PEGylation The purified proteins were concentrated to 2-3 mg / ml in 10k Amicon (Millipore) in 100 mM sodium phosphate buffer, pH 6.3, and 100 mM NaCl. For natural IL10, 20 kDa linear PEG (Sunbright® ME-200AL, NOF) was used, and for the inverse monomer, 40 kDa branched PEG (Sunbright® GL2-400AL3) was used to PEGylate the proteins at the N-terminus via aldehyde chemistry. A 5-fold molar excess of PEG and 10 mM sodium borohydride (NaCNBH3, Sigma) were added, and the PEGylation reaction was carried out at room temperature using end-to-end rotation for 24-48 hours. The progress of PEGylation was evaluated by analytical SEC. The PEGylated reaction mixture was diluted 10-fold and purified using a cation exchange column (SP HP, Cytiva) by stepwise elution with 20 mM sodium phosphate, pH 6.3, and 0.7 M NaCl to separate proteins from free PEG and NaCNBH3. The eluted proteins contained a mixture of non-PEGylated, mono-PEGylated, and multi-PEGylated protein species, which were further purified by size exclusion chromatography using a Superose 6 increase column (Cytiva) equilibrated in phosphate-buffered saline (PBS). The eluted peaks containing PEGylated proteins were pooled, concentrated to 0.5–2 mg / ml, and filtered sterile before use.

[0304] Surface plasmon resonance (Biacore) coupling All experiments were performed using a Biacore T200 instrument equipped with an anti-human capture (AHC)-CM5 chip (Cytiva) in 10 mM Hepes, 150 mM NaCl, 0.05% (v / v) polysorbate 20 (PS20), and 3 mM EDTA (HBS-EP+ buffer).

[0305] Human or mouse IL10RαFc (human: R&D systems # 9044-RI; mouse: Sino biologicals # 51298-M02H) was captured on the AHC surface. Since the IL10 analyte can be bivalent, the ligand capture density was set to 12 - 25 RU, and R max was limited to <8.5 RU (determined in another experiment), and the binding kinetics were measured under non-avid conditions. The IL10 analyte was injected at 0.74, 2.22, 6.66, 20, 60, 180 nM in high-performance mode (90 s association, 360 s dissociation), followed by surface regeneration with 3M MgCl2 (40 s, 30 μL / min). The sensograms subtracted by buffer were processed using Biacore T200 Evaluation Software and globally fitted using a 1:1 Langmuir binding model to extract the kinetic and affinity constants (ka, kd, KD). R max was in the range of 4 - 8.5 RU, showing a surface density that supports the true binding kinetics measurements in the absence of binding force.

[0306] The affinity of IL10 for IL10Rβ is weak (μM - mM) and is predicted to be undetectable under these conditions, so binding to human or mouse IL10Rβ has not been reported.

[0307] The affinity of the inverse monomer for IL10Ra showed a 3 - 6-fold decrease in K D compared to IL10 WT. See Tables 3 and 4 below.

[0308] (Table 3) Human molecules TIFF2026519565000068.tif42156

[0309] (Table 4) Mouse molecules TIFF2026519565000069.tif40151

[0310] Thermal stability measurement The proteins were formulated in PBS at approximately 1 mg / mL and aggregates were removed by 0.1 μm filtration. Each protein was loaded into four capillaries, and dynamic light scattering (DLS) and turbidity were measured at 1°C / min over a temperature gradient from 25°C to 95°C using a Prometheus Panta instrument. Unfolding data by differential scanning fluorescence quantification were not analyzed due to the lack of tryptophan in the IL10 molecule and insufficient signal. The data indicate that the manipulated molecules exhibit thermal stability comparable to IL10 WT, which begins to aggregate (DLS) at approximately 52°C. The data are shown in Figure 1.

[0311] Example 2: Evaluation of STAT3 activity This example describes a method for testing the activity of the polypeptides of the present disclosure, and the results of testing the activity of the polypeptides of the present disclosure.

[0312] pSTAT3 flow cytometry assay: Human Miltenyi MACSprep® PBMC isolation kit was used to purify PBMCs from healthy, non-smoking donor blood collected in a leukopenic system (LRS) chamber. Purified PBMCs (500,000 cells / well) were stimulated at 37°C for 20 minutes with IL10 proteins h_DR1061_AA (SEQ ID NO: 108) and h_SM0043_AA at concentrations ranging from 0.1 pM to 100 nM. The amino acid sequence of h_SM0043_AA is as follows: This is a wild-type human IL10 sequence expressed in Escherichia coli (E. coli) containing TIFF2026519565000070.tif17128. Cells were fixed at 37°C for 15 minutes using BD Phosflow® Fix Buffer I (BD Biosciences, catalog no. 557870). The cells were then washed and permeabilized overnight at -20°C using cooled BD Phosflow® Perm Buffer III (catalog no. 558050). The cells were washed to remove the permeabilization buffer and blocked at room temperature for 5 minutes using Human Trustain Fcx (Biolegend, catalog no. 422301) and mouse serum. The cells were then treated at room temperature for 1 hour with an antibody cocktail (listed in Table 14 below).

[0313] (Table 14) Antibody Cocktails TIFF2026519565000071.tif57146

[0314] After antibody staining, cells were washed, fixed, and flowed through a Cytek® Aurora Spectral flow cytometer. Data were analyzed using FlowJo software (www.flowjo.com). Various cell lines were gated using their respective lineage markers, and the geometric mean fluorescence intensity of pSTAT3 expression was calculated using FlowJo. The data are shown in Figures 2A and 2B. The presented data indicate that the inverse monomeric IL10 molecule (h_DR1060_AA) shows a decrease in pSTAT3 signaling in monocytes and CD8 T cells compared to the wild-type hIL10 molecule (h_SM0043_AA).

[0315] Example 3: Monocyte IL10 activity assay: Human PBMCs were isolated from LRS chambers obtained from non-smoking human donors by Ficoll® gradient purification. Human monocytes were purified from PBMCs by positive selection using human CD14 microbeads (Miltenyi Biotech, catalog no. 130-050-201) according to the manufacturer's protocol. Monocytes were treated with 1 ng / ml LPS (Millipore Sigma, catalog no. L2630) for 48 hours at 37°C in the presence of various concentrations of IL10 protein (0.05 pM to 200 nM). After treatment, cells were centrifuged at 400 g for 5 minutes and the supernatant was collected. The supernatant was evaluated for pro-inflammatory cytokines using the MSD kit (Meso Scale Discovery, catalog no. K151A9H-4), substantially according to the manufacturer's protocol. Data were graphed using Prism software (www.prismsoftware.com). The data are shown in Figure 3.

[0316] The data demonstrates that the inverse monomer IL-10 molecule (h_DR1061_AA) inhibits LPS-induced IL-1β secretion by monocytes, but its efficacy is 150 times lower than that of the naturally occurring dimer IL-10 molecule (h_SM0043_AA).

[0317] Example 4: CD8 T cell blast IL10 activity assay: Human PBMCs were isolated from LRS chambers obtained from non-smoking human donors by Ficoll gradient purification. Human CD8 T cells were purified from PBMCs by negative selection using the CD8+ T Cell Isolation Kit (Miltenyi Biotech-130-096-495), substantially following the manufacturer's protocol. Isolated CD8 T cells were activated using the Human T Cell Activation / Expansion Kit (Miltenyi Biotech, catalog no. 130-091-441) for 72 hours at 37°C in the presence of 100 pM human IL-2. After washing the activated CD8 T cells and magnetically removing the beads, the cells were treated with various concentrations of IL-10 protein (0.05 pM to 200 nM) for 72 hours at 37°C. After treatment, the cells were centrifuged at 400 g for 5 minutes and the supernatant was collected. The supernatant for cytokines IFNγ, granzyme A, and granzyme B was evaluated using the MSD kit (Meso Scale Discovery, catalog number K151AEL-4), substantially following the manufacturer's protocol. The data were graphed using Prism software. The data are shown in Figures 4A to 4C.

[0318] The data demonstrate that the inverse monomer IL10 molecule (h_DR1061_AA) does not induce the secretion of IFN-γ, granzyme A, or granzyme B by activated CD8 T cells, compared to the native hIL10 dimer (h-SM0043_AA).

[0319] Example 5: Mouse pSTAT3 flow cytometry assay: Spleens were removed from naive C57BL / 6 mice. Splenocytes were isolated by crushing the spleen in complete RPMI medium (Gibco RPMI 1640 + 10% FBS + 1% penicillin-streptomycin) using the plunger end of a syringe and filtering through a 70 μM strainer. The cells were centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 10 ml of ammonium chloride-potassium (ACK) lysis buffer (ThermoFisher) and allowed to stand at 37°C for 2 minutes. The cells were washed and resuspended in complete RPMI. Purified splenocytes (500,000 cells / well) were stimulated with IL10 protein at concentrations ranging from 0.1 pM to 100 nM for 20 minutes at 37°C. Cells were fixed at 37°C for 15 minutes using BD Phosflow® Fix Buffer I (BD Biosciences, catalog no. 557870). The cells were then washed and permeabilized overnight at -20°C using chilled BD Phosflow® Perm Buffer III (BD Biosciences, catalog no. 558050). The cells were washed to remove the permeabilization buffer and blocked at room temperature for 5 minutes using Mouse Trustain Fcx (Biolegend, catalog no. 101320). The cells were then treated at room temperature for 1 hour with an antibody cocktail (listed in Table 15 below).

[0320] (Table 15) Antibody Cocktails TIFF2026519565000072.tif43150

[0321] After antibody staining, cells were washed, fixed, and flowed through a Cytek® Aurora Spectral flow cytometer. Data were analyzed using FlowJo software. Various cell lines were gated using their respective lineage markers, and the geometric mean fluorescence intensity of pSTAT3 expression was calculated using FlowJo. Data from the mouse pSTAT3 flow cytometry assay are shown in Figures 5A and 5B.

[0322] The data showed that the mouse inverse monomer IL-10 molecule (m_WC161_AA) had lower EC induction of pSTAT3 in mouse bone marrow cells and CD8 T cells compared to the native dimer mRNA (m_DR768_AA). 50 To indicate that.

[0323] Example 6: Mouse splenocyte LPS assay: Splenes were removed from naive C57BL / 6 mice. Splenocytes were isolated by crushing the spleen in complete RPMI medium (Gibco RPMI 1640 medium + 10% FBS + 1% penicillin-streptomycin) using the plunger end of a syringe and filtering through a 70 μM strainer. The cells were centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 10 ml of ACK lysis buffer and allowed to stand at 37°C for 2 minutes. The cells were washed and resuspended in complete RPMI. Purified splenocytes (500,000 cells / well) were treated with 1 ng / ml LPS (Millipore Sigma, catalog number L2630) for 48 hours at 37°C in the presence of various concentrations of IL10 protein (0.1 pM to 100 nM). After treatment, the cells were centrifuged at 400 g for 5 minutes, and the supernatant was collected. The supernatant for the pro-inflammatory cytokines IL-6 and TNF-α was evaluated using the MSD kit (Meso Scale Discovery, catalog number K152A0H-2), substantially following the manufacturer's protocol. The data shown in Figures 6A and 6B were graphed using Prism software. The data indicate that the inverse monomeric mouse IL-10 molecule (m_WC161_AA) inhibits the secretion of IL-6 and TNF-α by mouse splenocytes, but with lower potency compared to the native dimeric IL-10 molecule (m_DR768_AA).

[0324] Example 7: Mouse CD8 T cell blast assay: Spleens were removed from naive C57BL / 6 mice. Splenocytes were isolated by crushing the spleen in complete RPMI medium (Gibco RPMI 1640 + 10% FBS + 1% penicillin-streptomycin) using the plunger end of a syringe and filtering through a 70 μM strainer. The cells were centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 10 ml of ACK lysis buffer and allowed to stand at 37°C for 2 minutes. The cells were washed and resuspended in complete RPMI. Mouse CD8 T cells were purified from PBMCs by negative selection using a kit (Miltenyi Biotech, catalog no. 130-104-075) according to the manufacturer's protocol. Isolated CD8 T cells were activated using the Mouse T Cell Activation and Expansion Kit (Miltenyi Biotech-130-093-627) for 72 hours at 37°C in the presence of 100 pM mouse IL-2. After washing the activated CD8 T cells and magnetically removing the beads, the cells were treated with various concentrations of IL10 protein (0.1 pM to 100 nM) in the presence of 100 pM mouse IL-2 at 37°C for 72 hours. After treatment, the cells were centrifuged at 400 g for 5 minutes and the supernatant was collected. The supernatant was evaluated for cytokines (granzyme B) using the MSD Kit (Meso Scale Discovery, catalog number K1523XR-2), substantially following the manufacturer's protocol. Data were graphed using Prism software. Data from the mouse CD8 T cell blast assay are shown in Figure 7.

[0325] The data show that the mouse monomeric IL10 molecule (m_WC161_AA) does not induce granzyme B secretion by activated CD8+ T cells compared to the native dimeric mRNA molecule (m_DR756_AA).

[0326] Example 8: Mouse CD8 T cell blast survival assay: Spleens were removed from naive C57BL / 6 mice. Splenocytes were isolated by crushing the spleen in complete RPMI medium (RPMI + 10% FBS + 1% penicillin-streptomycin) using the plunger end of a syringe and filtering through a 70 μM strainer. Cells were centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 10 ml of ACK lysis buffer and allowed to stand at 37°C for 2 minutes. Cells were washed and resuspended in complete RPMI. Mouse CD8 T cells were purified from PBMCs by negative selection using a kit (Miltenyi Biotech, catalog no. 130-104-075), substantially following the manufacturer's protocol. 200,000 CD8 T cells were added to each well of a 96-well plate pre-coated with 2.5 μg / ml anti-mouse CD3ε antibody (Biolegend 100302). Next, cells were treated with IL10 protein at concentrations ranging from 0.1 pM to 100 nM for 72 hours at 37°C in the presence of 5 μg / ml anti-mouse CD28 antibody (Biolegend 102102). The cells were then centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Cells were stained for annexin V and propidium iodide using the Annexin V Dead Cell Apoptosis Kit for Flow Cytometry (Invitrogen, catalog number V13241), substantially following the manufacturer's protocol. Data were analyzed using FlowJo software. After staining, cells were flowed through a Cytek Aurora Spectral flow cytometer. Data were analyzed using FlowJo software. Data were graphed using Prism software, as shown in Figure 8.

[0327] The data demonstrate that the mouse inverse monomer IL-10 molecule (m_WC161_AA), in contrast to the native dimer mRNA molecule (m_DR756_AA), does not enhance the survival of activated mouse CD8+ T cells.

[0328] Example 9: Serum protein concentration: Ten-week-old C57BL / 6J female mice (RRID:IMSR_JAX:000664) were subcutaneously injected with the indicated dose of IL-10 protein substantially produced as described in Example 1. Blood was collected via submandibular hemorrhage, and serum was prepared using serum microtubes (Sarstedt, product number 41.1378.005). Serum IL-10 concentrations were measured using an MSD kit (Meso Scale Discovery, K15069M). Concentrations were interpolated using a 4-parameter (4PL) model in GraphPad Prism 9.3.1 software. Concentrations are shown in Figure 9.

[0329] The data show that the PEG-modified mouse monomer IL-10 molecule (m_WC161_AAPEG) increased serum concentrations over a 72-hour period following a single-dose injection, compared to the natural dimer IL-10 molecule (m_DR756_AA_PEG).

[0330] Example 10: CD64 staining of monocyte cell surface: Human PBMCs were isolated from LRS chambers obtained from non-smoking human donors by Ficoll gradient purification. Human monocytes were purified from PBMCs by positive selection using human CD14 microbeads (Miltenyi Biotech, catalog no. 130-050-201) according to the manufacturer's protocol. Monocytes were treated with various concentrations of fused IL10 polypeptide (0.1 pM to 100 nM) at 37°C for 48 hours. After treatment, cells were centrifuged at 400 g for 5 minutes. Cells were then washed and blocked at 4°C for 15 minutes with Human Trustain Fcx (Biolegend, catalog no. 422301) and mouse serum. Cells were then treated with CD64 antibody (Biolegend, catalog no. 305006) at 4°C for 30 minutes. After antibody staining, cells were washed, fixed, and run through a Cytek Aurora Spectral flow cytometer. The data was analyzed using FlowJo software. The data shown in Figure 10 was graphed using Prism software.

[0331] The data show that the inverse monomer (h_DR1061_AA) induces significantly lower CD64 expression on the surface of monocytes compared to the native dimeric IL-10 molecule (h_SM0043_AA).

[0332] Example 11: Evaluation of inverse monomers in pSTAT3 flow cytometry assay: PBMCs were purified from healthy, non-smoking donor blood collected in a leukopenic system chamber (LRS) using the Human Miltenyi MACSprep PBMC Isolation Kit (Miltenyi Biotec, San Diego, CA), substantially in accordance with the manufacturer's instructions. Purified PBMCs (500,000 cells / well) were stimulated with IL10 protein at concentrations ranging from 0.1 pM to 100 nM for 20 minutes at 37°C. Cells were fixed with BD fix buffer I (BD Biosciences, catalog no. 557870) for 15 minutes at 37°C. Cells were then washed and permeabilized overnight at -20°C with cooled BD perm buffer III (BD Biosciences, catalog no. 558050). Cells were washed to remove the permeabilization buffer and blocked with Human Trustain Fcx (Biolegend, catalog no. 422301) and mouse serum for 5 minutes at room temperature. Next, the cells were treated with the antibody cocktail shown in Table 16 below at room temperature for 1 hour.

[0333] (Table 16) Antibody cocktail components TIFF2026519565000073.tif41152

[0334] After antibody staining, cells were washed, fixed, and flowed through a Cytek Aurora Spectral flow cytometer (Cytek Biosciences). Data were analyzed using FlowJo software (BD Biosciences, www.flowjo.com). Various cell lines were gated using their lineage markers, and the geometric mean fluorescence intensity of pSTAT3 expression was calculated using FlowJo. Data from this experiment are shown in Figures 12A and 12B of the attached drawings. As illustrated by the data shown in Figures 12A and 12B, monomeric IL10 molecules (h_DR1060_AA and h_DR1061_AA) exhibit reduced pSTAT3 signaling in monocytes and CD8 T cells compared to the native dimeric IL10 molecule (h_DR757_AA).

[0335] Example 12: In vivo evaluation of a DSS model for ulcerative colitis The therapeutic efficacy of the IL10 variant disclosed herein was evaluated in a mouse DSS model of ulcerative colitis. The mouse DSS-induced colitis model is widely used and shares many similarities with human ulcerative colitis. (Chassaing, B., et al. (2014) Current Protocols in Immunology 104:15.25.1-15.25.14).

[0336] The canonical sequence of the mature (without endogenous 18-amino acid signal peptide) wild-type mouse IL10 molecule (see UniProt P18893) is the following amino acid sequence: This is a 160-amino acid polypeptide having TIFF2026519565000074.tif12140. When referring to amino acid substitutions or deletions in the mouse IL10 variant molecule described herein, please refer to SEQ ID NO:141.

[0337] Table 17 below lists the PEG-treated mouse IL10 (mIL10) test samples used in the DSS test.

[0338] (Table 17) DSS test mouse specimens TIFF2026519565000075.tif26152

[0339] The amino acid sequences of the parent polypeptides of the m_DR756_AA_PEG and m_WC161_AA_PEG test samples are shown in Table 18 below.

[0340] (Table 18) Parent polypeptide of PEGylated DSS test product TIFF2026519565000076.tif72152

[0341] In short, the DSS study was conducted as follows: On days 0-6 of the study, female C57Bl / 6 mice (Jackson Laboratories) were administered 2.5% DSS in freely accessible drinking water. Mice given normal water served as negative disease controls. Mice were treated with either PBS, m_DR756_AA_PEG (3 or 10 micrograms subcutaneously every other day), or m_WC161_AA_PEG (10 or 30 micrograms subcutaneously every other day) according to Table 19 below.

[0342] (Table 19) DSS treatment group TIFF2026519565000077.tif47136

[0343] Mouse body weight was assessed throughout the study. Mouse body weight was measured three days before the start of DSS (Study Day 3), and after DSS administration and test substance treatment. Data regarding body weight for each treatment group are shown in Figure 13 of the attached diagram. Both mean body weight and mean % body weight + / - SEM are shown. The percentage of body weight was calculated as = (body weight on Study Day X - baseline body weight on Study Day 3) / (baseline body weight on Study Day 3). End-of-study body weight was assessed on Study Day 15. Mean % body weight + / - SEM on Day 15 is shown in Figure 14 of the attached diagram. Significance was determined by one-way ANOVA with Holm-Sidak multiple comparison test. ****p<0.0001

[0344] On day 15 of the study, terminal blood was collected by cardiac puncture and collected in a serum collection tube. Pre-vital and terminal serum were isolated by centrifugation of the coagulated blood at 10,000 RPM for 5 minutes. After centrifugation, the serum was collected and temporarily placed on ice before freezing for later analysis. Serum cytokines were evaluated using the Meso Scale Discovery Multiplex Kit (Meso Scale Discovery) substantially in accordance with the manufacturer's instructions.

[0345] On day 15 of the experiment, mice were sacrificed by CO2 asphyxiation. The colon was collected, feces were removed, and its length was measured. Data on colon length under various treatments are shown in Figure 15, reflecting the mean colon length + / - SEM. As shown by the data presented in Figure 15, m_DR756_AA_PEG, m_DR2677_AA_PEG, and m_DR2678_AA_PEG show evidence of protection against DSS-related outcomes and pathology.

[0346] Hematocrit levels were assessed on day 4 (D4) of the study. Blood samples collected by submandibular blood sampling on day 4 were placed in serum collection tubes and quantified using a Hemata Stat II hematocrit microcentrifuge (EKF Diagnostics, Inc.) substantially in accordance with the manufacturer's instructions. Data regarding hematocrit levels are shown in Figure 16 of the attached diagram, illustrating mean hematocrit + / - SEM. As illustrated, in m_DR756_AA_PEG, an estimated decrease in HCT may be observed on day 4.

[0347] Example 13: Evaluation of peritoneal exudative cell scavenger CD164 and CD64 expression over time. To collect peritoneal exudative cells, peritoneal lavage was performed on euthanized mice, and exudate was collected (peritoneal exudative cells, PEC). The peritoneal lavage fluid was centrifuged (1500 rpm, 5 min), the supernatant was removed, and the cell pellet was resuspended in 1 ml of PBS and kept on ice. Blood was collected in an EDTA-containing tube, transferred to a 5 ml tube, and erythrocytes were lysed using ACK lysis buffer (ThernoFisher #A1059201), then centrifuged (1500 rpm, 5 min). The supernatant was removed, and the cell pellet was resuspended in 1 ml of PBS. Peritoneal exudative cells were collected over time, and CD163 or CD64 was detected by flow cytometry. Median and mean median fluorescence intensity + / - SEM values ​​for individual cells are also shown. APC = antigen-presenting cell. As illustrated in Figure 17, treatment with a PEGylated wild-type mouse IL10 substitute molecule (m_DR756_AA_PEG) resulted in CD163 upregulation on macrophages. CD64 expression was also evaluated. The results are shown in Figure 18.

[0348] Example 14: Evaluation of peritoneal exudative cell scavenger CD164 and CD64 expression over time. Serum was collected at various time points during the study, and serum cytokines (IFNγ, IL10, IL-1B, IL6, TNF-α, IL-4, IL-5, IP10, IL17A / F, MIP1α) were measured. Serum cytokines were evaluated using the Meso Scale Discovery multiplex kit (Meso Scale Discovery) substantially in accordance with the manufacturer's instructions. Data for the various treatment groups are shown in Figures 19 and 20 of the attached drawings, reflecting mean analyte concentrations (pg / mL) + / - SEM. As illustrated by the data present in Figures 19 and 20, treatment with the inverse monomer (m_WC161_AA_PEG) is generally associated with a decrease in systemic IFNγ and TNFα compared to the wild-type molecule (m_DR756_AA_PEG). Treatment with m_DR756_AA_PEG is associated with an increase in serum IP-10 and MIP1α. Treatment with the inverse monomer (m_WC161_AA_PEG) is associated with elevated serum IL-4 and IL-5 (Th2 cytokines).

[0349] After assessing colon length, isolated colons were placed in formalin at room temperature for 24 hours, then processed using a tissue processing device according to a standardized procedure and embedded in FFPE blocks. The FFPE blocks were sectioned to a thickness of 5 μm and placed on charged glass slides.

[0350] Multiplex immunofluorescence analysis was performed on collected tissues to check for the presence of the following markers: RORγ, CD11b, and CD3e. The primary antibodies used for immunohistochemical analysis were as follows: anti-mouse: RORγ (Abcam, catalog ab207802), active concentration 1:750; anti-CD11b (Abcam, catalog ab216445), active concentration 1:3000; and anti-CD3e (Thermo / Invitrogen, catalog MA1-90582), active concentration 1:200. All antibodies were incubated at room temperature for 60 minutes.

[0351] Slides were stained with antibodies using the Leica Bond Rx (Leica Biosystems) automated staining system. The automated staining system was programmed to perform the following steps: (a) heat-induced epitope recovery (HIER) and deparaffinization for 20 minutes at 97°C in the presence of BOND-PRIME Epitope Retrieval Solution 2 (ER2) (Leica Biosystems); (b) peroxidase quenching; (c) incubation with primary antibody for 60 minutes; (d) incubation with secondary antibody polymer for 30 minutes; and (e) detection fluorophore for 10 minutes at room temperature. Fluorophores used to visualize the signal included Opal 520, Opal 620, and Opal 690 fluorophores (Akoya Biosciences, Marlborough MA).

[0352] For labeling, staining was performed sequentially by including a second antigen recovery step between the two steps to remove any unbound reagents from the preceding marker using BOND-PRIME Epitope Retrieval Solution 1 (ER1) (Leica Biosystems). Appropriate cross-reactivity controls were included for quality control.

[0353] After fluorescence staining was completed, the tissue was counterstained with Akoya spectral DAPI (Akoya Biosciences) and covered with a coverslip using ProLong® Gold Antifade Mountant (ThermoFisher Scientifid / Invitrogen, catalog number: P36930) aqueous mounting medium. Whole slide scans and signal quantification were performed using the Akoya Vectra multispectral imaging system (Akoya Biosciences) and the Akoya Vectra InForm analysis software suite (Akoya Biosciences). Digital quantification of specific staining signals was performed, and data were reconciled and aggregated from all images using the Akoya Phenoptr Reports software package (Akoya Biosciences).

[0354] Histological evaluation was performed for colonic epithelial damage. The presence of intact epithelial layers was assessed using Visiopharm software (Visiopharm A / S, DK 2970-Horsholm, Denmark) on whole-slide scanned hematoxylin and eosin (H&E) stained mouse colon sections. Data from various treatment groups are shown in Figure 21 of the attached diagram, reflecting mean percentage colonic epithelial damage ± SEM.

[0355] The presence of CD11b and CD4+ RorG T cells was evaluated using inForm image analysis software (Akoya Biosciences) on multiplex immunofluorescence-stained mouse colon sections scanned as whole slides. Data from various treatment groups are shown in Figure 22 of the attached diagram, with mean cell count / mm². 2 ±SEM is reflected.

[0356] In summary, data obtained in a DSS model of ulcerative colitis indicate that the inverse monomer (m_WC161_AA_PEG) (a) provides evidence of protection against DSS-related outcomes and pathology, (b) provides protection against DSS-related epithelial damage, and (c) reduces myeloid and Th17 infiltration in the intestinal mucosa.

[0357] Example 15: In vivo evaluation of pharmacokinetic parameters: The pharmacokinetic parameters of the m_DR756_AA_PEG and m_WC161_AA_PEG test substances were evaluated in an in vivo study in C57Bl / 6 mice. Briefly, on day 0 (D0), female C57Bl / 6 mice received a single subcutaneous dose of 10 micrograms of m_DR756_AA_PEG or 10, 30, or 90 micrograms of m_WC161_AA_PEG. At various time points, living blood samples were collected in serum collection tubes by submandibular blood collection. Terminal blood was collected by cardiac puncture and collected in serum collection tubes. Living and terminal serum were isolated by centrifugation of the coagulated blood at 10,000 RPM for 5 minutes. After centrifugation, serum was collected and temporarily placed on ice before freezing for later analysis. Test substances in serum were quantified using the mouse IL-10 MSD U-plex kit (Meso Scale Discovery) substantially in accordance with the manufacturer's instructions. Each individual test sample was used as a standard for detection.

[0358] Serum was collected over time, and the serum concentration of the test substance was quantified over time. Data reflecting the average test substance concentration (ng / mL) + / - SEM are shown in Figure 23 of the attached diagram. As illustrated in Figure 23, m_WC161_AA_PEG showed improved systemic exposure compared to m_DR756_AA_PEG. Furthermore, the inverse monomer (m_WC161_AA_PEG) showed high sustained exposure after multiple administrations.

[0359] Example 16: Evaluation of pSTAT3 induction in bone marrow cells over time The effect of the test material on its ability to induce pSTAT3 over time in bone marrow cells was evaluated as follows: For pSTAT or surface immunophenotyping flow cytometry, viable or terminal whole blood from the experiments described in the above examples was collected in EDTA tubes. 100-200 μL of whole blood collected in EDTA tubes was transferred to a 96-well deep-well plate. BD Phosflow lysis / fixation buffer (catalog no. 558049) was added to a total volume of 2 ml, and the plate was incubated at 37°C for 10 minutes. The cells were centrifuged at 600 g for 7 minutes, the supernatant was removed, and the cells were washed twice with 1 ml of PBS / 2% FBS per well. The cell pellet was vortexed and resuspended, 1 ml of pre-chilled BD Perm buffer III (catalog no. 558050) was added, and the cells were stored at -80°C. After the completion of viable sample collection, the cells were thawed and washed three times to remove the permeabilization buffer. Cells were blocked on ice for 10 minutes with 1:25 TruStain FcX (Biolegend #101320) and 1:50 rat serum, and then incubated with the antibody cocktail at 4°C for 30 minutes (see Table 12 below). Cells were washed twice, resuspended in PBS / 2% FBS, and run through a Cytek Aurora Spectral cytometer. Data were analyzed using Cell Engine. Cells were gated using strain markers, and median fluorescence intensities for pSTAT 1 and pSTAT 3 were calculated.

[0360] (Table 20) pSTAT staining panel TIFF2026519565000078.tif47149

[0361] To evaluate the time course of pSTAT3 induction in bone marrow cells, peripheral blood was collected over time and pSTAT3 was detected by flow cytometry. The data are shown in Figure 24 of the attached diagram. As illustrated in Figure 24, the inverse monomer (m_WC161_AA_PEG) showed evidence of pSTAT3 induction in PBMCs at 48 and 72 hours post-administration.

[0362] array The nucleic acid sequences encoding the inverse monomers in Table 6 are shown in Table 5 below.

[0363] (Table 5) Human reverse monomer nucleic acid sequences TIFF2026519565000079.tif172147TIFF2026519565000080.tif213147

[0364] (Table 6) Human reverse monomer amino acid sequences TIFF2026519565000081.tif144147

[0365] (Table 7) Human dimerization Reverse monomer nucleic acid sequence TIFF2026519565000082.tif172147TIFF2026519565000083.tif176147TIFF2026519565000084.tif172147

[0366] (Table 8) Human dimerization Inverse monomer amino acid sequence TIFF2026519565000085.tif165147

[0367] (Table 9) Mouse inverse monomer nucleic acid sequences TIFF2026519565000086.tif143147

[0368] (Table 10) Mouse reverse monomer amino acid sequences TIFF2026519565000087.tif57147

[0369] Table 10B shows additional mouse inverse dimers of the present disclosure. Any substitutions referred to herein are based on the mature mIL10 canonical reference sequence: The molecules are numbered according to TIFF2026519565000088.tif13151. Substantially in accordance with the instruction in the examples and the data shown in Table 10A, the binding specificity of the mouse inverse monomers shown in Table 10A to the immobilized mIL10Ra receptor subunit conjugated to each arm of the immobilized Fc was confirmed by surface plasmon resonance. As described in the examples, the affinity of IL10 to the IL10Rb subunit in the absence of IL10 is extremely low, making measurement by SPR difficult. Therefore, the molecules were evaluated by ELISA to confirm binding to both IL10Ra and IL10Rb. The data show that some of these inverse monomers showed greater binding affinity to the IL10Ra receptor than the WC131 control molecule described below. Based on the sequence homology between mouse and human molecules, the amino acid substitutions in the mouse inverse monomers described in Tables 10 and 10B can be incorporated into the human inverse monomer of Formula 1. In some embodiments, the disclosure provides human inverse monomers comprising one or more amino acid substitutions at the positions substituted in homologous mouse inverse monomers of Table 10 or Table 10B.

[0370] (Table 10A) mMIL10Ra SPR coupling data TIFF2026519565000089.tif102140

[0371] (Table 10B) Mouse inverse monomer sequence TIFF2026519565000090.tif192145TIFF2026519565000091.tif192145TIFF2026519565 000092.tif192145TIFF2026519565000093.tif192145TIFF2026519565000094.tif47145

[0372] (Table 11) Mouse dimerized inverse monomer nucleic acid sequences TIFF2026519565000095.tif172147

[0373] (Table 12) Mouse dimerized inverse monomer amino acid sequences TIFF2026519565000096.tif61147

Claims

1. Formula 1: [A]-L1x-[B]-L2-[C]-L3-[D]-L4y-[E]-L5-[F] (1) A polypeptide comprising the amino acid sequence, During the ceremony, x and y are chosen independently from 0 (non-existent) or 1 (existent), [A] is the amino acid sequence In comparison, polypeptides having 0, 1, or 2 amino acid substitutions, x = 0 (no linker, L1 does not exist), or x = 1, and L1 contains a polypeptide linker of 1 to 5 amino acids. [B] is the amino acid sequence In comparison, polypeptides having 0, 1, or 2 amino acid substitutions, L2 contains a linker of 10-25 amino acids. [C] is the amino acid sequence In comparison, polypeptides having 0, 1, or 2 amino acid substitutions, L3 contains a linker of 4 to 11 amino acids. [D] is the amino acid sequence In comparison, polypeptides having 0, 1, or 2 amino acid substitutions, y = 0 (L4 does not exist), or y=1, and L4 contains a polypeptide linker of 1-5 amino acids. [E] is the amino acid sequence In comparison, polypeptides having 0, 1, or 2 amino acid substitutions, L5 contains a linker with 1 to 7 amino acids. [F] is the amino acid sequence In comparison, polypeptides having 0, 1, or 2 amino acid substitutions, The aforementioned polypeptide.

2. The polypeptide according to claim 1, wherein L1, L2, L3, L4 and / or L5 comprises a GS-linker.

3. The polypeptide according to claim 1 or 2, wherein L1 is the amino acid glutamine (Q).

4. L2 is the amino acid sequence A polypeptide according to any one of claims 1 to 3, wherein the polypeptide is a polypeptide having the following characteristics.

5. The polypeptide according to any one of claims 1 to 4, wherein L3 is a polypeptide having the amino acid sequence QLDNLLL (SEQ ID NO: 8).

6. The polypeptide according to any one of claims 1 to 5, wherein L4 is the amino acid glycine ("G") or comprises the amino acid sequence GY.

7. The polypeptide according to any one of claims 1 to 6, wherein L5 is a polypeptide having the amino acid sequence QDPD (SEQ ID NO:9).

8. A polypeptide according to any one of claims 1 to 7, comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO:10 or SEQ ID NO:

11.

9. amino acid sequence The polypeptide according to claim 8, comprising:

10. amino acid sequence The polypeptide according to claim 8, comprising:

11. A polypeptide according to any one of claims 1 to 8, comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to an inverted monomer of hIL10 selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:

98.

12. Formula (2): [Monomer 1]-Linker x - [Monomer 2] (2) A polypeptide comprising, In the formula, monomer 1 and monomer 2 each independently contain polypeptides selected from the polypeptides described in claim 1, and x = 0 (no linker present) or 1 (a linker present). The aforementioned polypeptide.

13. The polypeptide according to claim 12, wherein monomer 1 and monomer 2 each independently contain a polypeptide having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:

98.

14. The polypeptide according to claim 13, wherein monomer 1 and monomer 2 each comprise a polypeptide containing the amino acid sequence of SEQ ID NO:

10.

15. The polypeptide according to claim 13, wherein monomer 1 and monomer 2 each comprise a polypeptide having the amino acid sequence of SEQ ID NO:

11.

16. The polypeptide according to any one of claims 12 to 15, wherein x is 1 (a linker is present) and the linker includes a GS-linker.

17. The polypeptide according to any one of claims 12 to 15, wherein x is 0 (no linker exists).

18. The polypeptide of formula (2) above is converted in the order from amino to carboxyl, (a) Below: (i) A first polypeptide having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide selected from the group consisting of amino acid residues 117-158 (SEQ ID NO:12), 117-159 (SEQ ID NO:13), and 117-160 (SEQ ID NO:14) of human IL-10, numbered according to mature wild-type human IL-10 (SEQ ID NO:20), (ii) The first linker where x = 0 (no linker exists) or 1 (a linker exists) x and, (iii) Numbered according to mature wild-type hIL10 (SEQ ID NO:20), amino acid residues 1-160 of human IL-10 (SEQ ID NO:20), amino acid residues 2-160 of human IL-10 (SEQ ID NO:36), amino acid residues 3-160 of human IL-10 (SEQ ID NO:37), amino acid residues 4-160 of human IL-10 (SEQ ID NO:38), amino acid residues 5-160 of human IL-10 (SEQ ID NO:39), amino acid residues 6-160 of human IL-10 (SEQ ID NO:40), amino acid residues 7-160 of human IL-10 (SEQ ID NO:41), amino acid residues 8-160 of human IL-10 (SEQ ID NO:42), amino acid residues 9-160 of human IL-10 (SEQ ID NO:43), amino acid residues 10-160 of human IL-10 (SEQ ID NO: A second polypeptide comprising amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 11-160 of human IL-10 (SEQ ID NO:44) and human IL-10 (SEQ ID NO:45), and Includes monomer 1, (b) A second linker where y = 0 (no linker exists) or 1 (a linker exists). y , and (c) Numbered according to mature human IL-10 (SEQ ID NO:20), amino acid residues 1-116 of human IL-10 (SEQ ID NO:15), amino acid residues 2-116 of human IL-10 (SEQ ID NO:16), amino acid residues 3-116 of human IL-10 (SEQ ID NO:17), amino acid residues 4-116 of human IL-10 (SEQ ID NO:18), amino acid residues 5-116 of human IL-10 (SEQ ID NO:19), amino acid residues 6-116 of human IL-10 (SEQ ID NO:29), amino acid residues 7-116 of human IL-10 (SEQ ID NO:30), amino acid residues 8-116 of human IL-10 (SEQ ID NO:31), amino acid residues 9-116 of human IL-10 (SEQ ID NO:32), amino acid residues 10-116 of human IL-10 (SEQ ID Monomer 2 contains a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polypeptide sequence selected from the group consisting of amino acid residues 11-116 of human IL-10 (SEQ ID NO:33), and human IL-10 (SEQ ID NO:34). The polypeptide according to claim 12, comprising:

19. The polypeptide of formula (2) above is converted in the order from amino to carboxyl, (a) Below: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 (SEQ ID NO:12), 117-159 (SEQ ID NO:13), and 117-160 (SEQ ID NO:14) of human IL-10, numbered according to mature human IL-10 (SEQ ID NO:20), (ii) The first linker where x = 0 (no linker exists) or 1 (a linker exists) x , (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1 to 116 (SEQ ID NO: 15) of human IL-10. Includes monomer 1, (b) A second linker where y = 0 (no linker exists) or 1 (a linker exists). y , (c) Below: (i) A first polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 117-158 (SEQ ID NO:12), amino acid residues 117-159 (SEQ ID NO:13), and amino acid residues 117-160 (SEQ ID NO:14) of human IL-10, numbered according to mature human IL-10 (SEQ ID NO:20), (ii) A third linker where z = 0 (no linker exists) or 1 (a linker exists) z and, (iii) A second polypeptide comprising an amino acid sequence selected from the group consisting of amino acid residues 1 to 116 (SEQ ID NO: 15) of human IL-10, Includes monomer 2 The polypeptide according to claim 12, comprising:

20. The polypeptide according to claim 12, comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to an hIL10 reverse monomer selected from the group consisting of SEQ ID NO: 46 to 51.

21. A polypeptide according to any one of claims 1 to 20, which is PEGylated.

22. The polypeptide according to claim 21, wherein the PEG molecule is linear or branched and has a molecular weight of about 10 kD to about 80 kD.

23. The polypeptide according to claim 22, wherein the PEG molecule is a 40 kD branched PEG molecule containing two 20 kD arms.

24. The polypeptide according to any one of claims 21 to 23, wherein a PEG molecule is covalently bonded to the N-terminus of the polypeptide.

25. A polypeptide according to any one of claims 1 to 24, which exhibits cell type biasing activity compared to wild-type IL10 species derived from inverse IL10 monomers.

26. The polypeptide according to claim 25, which (a) exhibits a significant level of at least one anti-inflammatory property of wild-type IL10, and (b) exhibits a significantly reduced level of at least one pro-inflammatory property of wild-type IL10.

27. The polypeptide according to claim 26, wherein the at least one anti-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IL1b, TNFa, or IL6 in bone marrow cells.

28. The polypeptide according to claim 27, wherein the at least one pro-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IFNγ, granzyme A, or granzyme B in T cells.

29. The polypeptide according to claim 27, wherein the at least one anti-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IL1b, TNFa, or IL6 in bone marrow cells, and the at least one pro-inflammatory property is selected from the group consisting of suppression of the expression or secretion of IFNγ, granzyme A, or granzyme B in T cells.

30. The polypeptide according to claim 25, wherein the cell type biasing activity is the production of phospho-STAT3.

31. The pSTAT3 E of the polypeptide max However, in bone marrow cells, wild-type hIL10 pSTAT3 E max The polypeptide according to claim 30, wherein the amount is more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, or more than 70%.

32. The pSTAT3 E in lymphocytes max is less than 70%, less than 60%, less than 50%, less than 40% or less than 30% of the pSTAT3 E of wild-type hIL10 in lymphocytes max The polypeptide according to claim 31, wherein the polypeptide is less than 70%, less than 60%, less than 50%, less than 40% or less than 30% of the pSTAT3 E of wild-type hIL10 in lymphocytes

33. (a) pSTAT3 E of the polypeptide max However, in bone marrow cells, wild-type hIL10 pSTAT3 E max (b) the pSTAT3 E in lymphocytes is greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70% max However, pSTAT3 E of wild-type hIL10 in lymphocytes max The polypeptide according to claim 30, wherein the amount is less than 70%, less than 60%, less than 50%, less than 40%, or less than 30%.

34. A nucleic acid sequence encoding the polypeptide according to any one of claims 1 to 33.

35. A recombinant vector comprising the nucleic acid sequence according to claim 34, functionally linked to one or more expression control sequences.

36. Recombinant cells transformed with the recombinant vector according to claim 35.

37. A method for producing a polypeptide according to any one of claims 1 to 33, comprising the steps of (a) culturing the host cells according to claim 36 under conditions suitable for the expression of the polypeptide, and (b) recovering the polypeptide from the host cell culture.

38. The method according to claim 37, wherein the host cell is a mammalian host cell.

39. The method according to claim 37, wherein the host cell is a bacterial cell.

40. A composition comprising a polypeptide according to any one of claims 1 to 33, a nucleic acid sequence according to claim 34, or a recombinant vector according to claim 35, and one or more pharmaceutically acceptable salts, excipients and / or diluents.

41. A method for treating a mammalian subject suffering from a disease, disorder or condition, comprising the step of administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1 to 33, or a composition according to claim 40.

42. The method according to claim 41, wherein the disease, disorder, or condition is an autoimmune disease, disorder, or condition.

43. The aforementioned autoimmune diseases, disorders, or conditions include ulcerative colitis, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes including type 1 or type 2 diabetes, inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, and SEA syndrome (Seronegativity Enthesopathy Arthropathy). The method according to claim 42, selected from the group consisting of juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarthritis, polyarthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteritis arthritis, reactive arthritis, Reiter's syndrome, and SEA syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome).

44. The method according to claim 41, wherein the disease, disorder, or condition is cancer associated with chronic inflammation.

45. The method according to any one of claims 41 to 44, wherein the treatment prevents the progression of the disease, disorder, or condition.

46. The method according to any one of claims 41 to 44, wherein the treatment improves one or more symptoms of the disease, disorder, or condition.

47. A method for preventing a disease, disorder or condition in a mammal at risk of developing the disease, disorder or condition, comprising the step of administering a preventively effective amount of the composition according to claim 40 to the subject before the onset of symptoms of the disease, disorder or condition.

48. The method according to claim 47, wherein the disease, disorder, or condition is cancer associated with chronic inflammation.

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