Fusion IL10 polypeptide

Abstract

This disclosure relates, in general terms, to compositions and methods for modulating interleukin-10 (IL10)-mediated signaling. In particular, this disclosure provides a fusion IL10 polypeptide comprising two IL10 monomers. Also provided are compositions and methods useful for producing such a fusion IL10 polypeptide, as well as methods for modulating IL10-mediated signaling and / or treating conditions associated with disruption of IL10-mediated signaling. TIFF2026521391000111.tif103150

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 506,000, filed on 2 June 2023, the disclosure of which is incorporated herein by reference in its entirety for any purpose. [Background technology]

[0002] Background of this disclosure 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 designation TIFF2026521391000002.tif64145 (UniProt reference number Q13651). Residues 22-235 of SEQ ID NO:1 (amino acids 1-214 of the mature hIL10Rβ protein) correspond to the extracellular domain (ECD), residues 236-256 of SEQ ID NO:1 (amino acids 215-235 of the mature hIL10Rβ protein) correspond to the transmembrane domain (TM), and residues 257-578 of SEQ ID NO:1 (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, TIFF2026521391000003.tif37145 (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, TIFF2026521391000004.tif37145.

[0009] mIL10Rβ 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 IL10R2 and the ternary complex of IL10 / sIL10R1 / sIL10R2, 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 of hIL10 with 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 the rate-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 that are phosphorylated in response to hIL-10 and required for hIL-10 signaling.

[0012] Homodimerization of STAT3 leads to its release from the receptor and the translocation of phosphorylated STAT homodimers into the nucleus, where the phosphorylated STAT 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 has 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 Literature 7).

[0013] The expression of hIL10Rα receptor subunits and IL10Rβ 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 IL10Rβ 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.

[0014] 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.

[0015] Saxton et al. have described the interaction between IL10 and the IL10Rβ 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]

[0016] [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]

[0017] Summary of this disclosure This disclosure provides compositions useful for modulating interleukin-10 (IL10)-mediated signaling. In particular, this disclosure provides a fusion IL10 polypeptide comprising hIL10A and hIL10B, which are two IL10 monomers independently selected from the group consisting of wild-type hIL10 (SEQ ID NO:4) and hIL10 mutein, and which can be linked with or without a linker. Also provided are compositions and methods useful for producing such a fusion IL10 polypeptide, as well as methods for modulating IL10-mediated signaling and methods for treating conditions associated with perturbations of IL10-mediated signaling.

[0018] In one aspect, this disclosure is based on the following formula: (hIL10A)-L n -(hIL10B) We provide a fused human IL10 (hIL10) polypeptide containing the polypeptide, During the ceremony, (a) hIL10A and hIL10B are human IL10 (hIL10) sequences independently selected from the group consisting of wild-type hIL10 (SEQ ID NO:4) and hIL10 mutein, and each hIL10 mutein independently contains one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96 and R104 of SEQ ID NO:4, and optionally hIL10A has amino-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues compared to SEQ ID NO:4, and / or hIL10B has amino-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues compared to SEQ ID NO:4. (b) L is an amino acid linker with a length of 1 to 30 amino acids, and (c) n = 0 (does not exist) or 1 (exists).

[0019] In some embodiments of the fused hIL10 polypeptide, (a) The amino acid substitution at position T100 is selected from the group consisting of T100L, T100D, T100V, T100E, T100A, T100R, T100N, T100Q, T100E, T100I, T100K, T100M, and T100S. (b) The amino acid substitution at position H14 is selected from the group consisting of H14C, H14F, H14P, H14W, H14G, H14A, H14D, H14E, H14I, H14K, H14L, H14M, H14N, H14Q, H14R, H14S, H14T, H14Y, and H14V. (c) The amino acid substitution at position N18 is selected from the group consisting of N18Y, N18F, N18A, N18D, N18E, N18L, N18V, N18S, N18T, N18I, N18V, N18M, N18R, N18K, and N18H. (d) The amino acid substitution at position N21 is selected from the group consisting of N21A, N21R, N21Q, N21H, N21K, N21S, N21V, N21I, N21L, N21M, N21T, N21C, N21D, and N21E. (e) The amino acid substitution at position M22 is selected from the group consisting of M22A, M22V, M22I, M22L, M22N, M22D, M22S, M22T, M22W, and M22Q. (f) The amino acid substitution at position R24 is selected from the group consisting of R24E, R24D, R24N, R24Q, R24A, R24S, and R24T. (g) The amino acid substitution at position D25 is selected from the group consisting of D25A, D25N, D25H, D25I, D25K, D25L, D25P, D25Q, and D25V. (h) The amino acid substitution at position D28 is selected from the group consisting of D28A, D28E, D28L, D28V, D28S, D28T, D28I, D28V, D28M, D28H, D28K, and D28R. (i) The amino acid substitution at position R32 is selected from the group consisting of R32A, R32D, R32E, R32L, R32V, R32S, R32T, R32I, R32V, R32M, R32N, R32Q, R32G, R32C, R32P, R32F, R32Y, and R32H. (j) The amino acid substitution at position E74 is selected from the group consisting of E74A, E74D, E74L, E74V, E74S, E74T, E74I, E74V, E74M, E74H, E74K, and E74R. (k) The amino acid substitution at position H90 is selected from the group consisting of H90A, H90D, H90E, H90I, H90K, H90L, H90M, H90N, H90Q, H90R, H90S, H90T, H90Y, and H90V. (l) The amino acid substitution at position N92 is selected from the group consisting of N92D, N92Q, N92E, N92H, N92K, N92S, N92V, N92I, N92L, N92M, N92T, and N92A. The amino acid substitution at position (m)S93 is selected from the group consisting of S93E, S93A, S93R, S93N, S93D, S93Q, S93E, S93I, S93L, S93K, S93M, S93G, and S93V. (n) The amino acid substitution at position E96 is selected from the group consisting of E96C, E96F, E96Y, E96W, E96A, E96N, E96D, E96Q, E96H, E96K and E96S, and (o) The amino acid substitution at position R104 is selected from the group consisting of R104A, R104W, R104Y, R104F, R104H, R104D, R104E, R104N, R104Q, R104S, R104T, R104I, R104L, R104V, and R104M.

[0020] In some embodiments, at least one of hIL10A and hIL10B is hIL10 mutaine. In certain embodiments, both hIL10A and hIL10B are hIL10 mutaine. In certain embodiments, hIL10A and hIL10B are the same. In certain embodiments, hIL10A and hIL10B are different.

[0021] In some embodiments, n=0 (non-existent). In some embodiments, n=1 (existent), and L is a polypeptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, L is a GS linker. In certain embodiments, the GS linker contains the sequence GGGSGSGSGSG (SEQ ID NO: 19).

[0022] In some embodiments, hIL10A and hIL10B are independently selected from wild-type hIL10 (SEQ ID NO: 4) or from IL10 muteins containing amino acid substitutions selected from the group consisting of N21D, N21E, N21K, M22A, M22S, M22T, M22D, M22W, R24E, D25K, E96K, E96Q, T100E, T100C, and T100L. In certain embodiments, hIL10A and / or hIL10B contain the amino acid substitution T100L. In some embodiments, hIL10A and / or hIL10B contain the amino acid substitution M22A or M22S. In some embodiments, hIL10A and / or hIL10B contain the amino acid substitution E96Q. In some embodiments, hIL10A and / or hIL10B contain the amino acid substitution R24E. In some embodiments, hIL10A and / or hIL10B contain the amino acid substitution D25K. In some embodiments, hIL10A and / or hIL10B contain the amino acid substitution N21K.

[0023] In some embodiments of the fused hIL10 polypeptide, hIL10A and / or hIL10B are, respectively, SEQ ID A polypeptide having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a polypeptide selected from the group consisting of NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, wherein either or both of hIL10A and hIL10B are SEQ ID Compared to the sequence of NO:4, it has N-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In certain embodiments, hIL10A and hIL10B are the same. In certain embodiments, hIL10A and hIL10B are different. In certain embodiments, hIL10A and hIL10B each contain amino acid substitutions selected from the group consisting of N21D, N21E, N21K, M22A, M22S, M22T, M22D, M22W, R24E, D25K, E96K, E96Q, T100E, T100C, and T100L.

[0024] In certain embodiments, hIL10A is a polypeptide selected from the group consisting of SEQ ID NO: 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, and hIL10B is a polypeptide selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, and 156.

[0025] In some embodiments, both hIL10A and hIL10B are hIL10 mutaines containing the amino acid substitution T100L. In certain embodiments, both hIL10A and hIL10B are hIL10 mutaines containing the amino acid substitution T100L, where hIL10A is a polypeptide having 100% sequence identity to SEQ ID NO:204, and hIL10B is a polypeptide having 100% sequence identity to SEQ ID NO:15.

[0026] In some embodiments, the fused hIL10 polypeptide is SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 119, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, A polypeptide comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a polypeptide selected from the group consisting of 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, and 186.

[0027] In some embodiments, the fused hIL10 polypeptide contains an amino acid sequence that has 100% sequence identity with a polypeptide selected from the group consisting of SEQ ID NO: 44, 123, 124, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, and 186.

[0028] In some embodiments, the fusion hIL10 polypeptide contains an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with a polypeptide selected from the group consisting of SEQ ID NO:44, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:170, and SEQ ID NO:185. In certain embodiments, the fusion hIL10 polypeptide contains a polypeptide having 100% identity with the amino acid sequence of SEQ ID NO:44. In certain embodiments, the fusion hIL10 polypeptide contains a polypeptide having 100% identity with the amino acid sequence of SEQ ID NO:185.

[0029] In some embodiments, the fused hIL10 polypeptide is modified to extend its half-life in vivo. In certain embodiments, the modification for extending the half-life in vivo is selected from the group consisting of PEGylation, acylation, albumination, or conjugation to an Fc polypeptide. In certain embodiments, the modification for extending the half-life in vivo is acylation. In certain embodiments, the modification for extending the half-life in vivo is conjugation to an Fc polypeptide. In certain embodiments, the Fc polypeptide is an Fc domain derived from hIgG1, hIgG2, hIgG3, or hIgG4, or a variant thereof. In certain embodiments, the Fc polypeptide contains a sequence modified from the wild-type Fc polypeptide sequence to reduce effector function. In some embodiments, the Fc polypeptide contains one or more amino acid substitutions or deletions to promote heterodimerization.

[0030] In some embodiments, the fused hIL10 polypeptide is formula #1: IL10FP-L1 a -UH1-Fc1 [1] The first polypeptide and formula #2: UH2-Fc2 [2] A heterodimer Fc molecule containing a second polypeptide, During the ceremony, hIL10FP is the fused hIL10 polypeptide of the present disclosure (e.g., formula (hIL10A)-L n -(hIL10B) is a fused hIL10 polypeptide, L1 is the linker, and a is selected independently of 0 (non-existent) or 1 (existent). UH1 and UH2 are upper hinge domains of human immunoglobulins, independently selected from the group consisting of the upper hinge domains of IgG1, IgG2, IgG3, and IgG4, and optionally contain the amino acid substitution C220S (EU numbering). Fc1 is a polypeptide comprising a lower hinge, CH2 domain, and CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and contains one or more amino acid substitutions that promote heterodimerization with Fc2. Fc2 is a polypeptide comprising a lower hinge, CH2 domain, and CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and contains one or more amino acid substitutions that promote heterodimerization with Fc1. Optionally, the polypeptide of formula [1] and the polypeptide of formula [2] are linked by at least one interchain disulfide bond.

[0031] In some embodiments, the modification for extending the half-life in vivo is PEGylation. In certain embodiments, PEG is a linear or branched polyethylene glycol molecule having a molecular weight 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. In certain embodiments, PEG is linear. In certain embodiments, PEG is branched. In some embodiments, PEG is a 40 kD branched PEG molecule containing two 20 kD arms. In certain embodiments, PEG is optionally covalently bonded to the N-terminus of a polypeptide via a linker.

[0032] In certain embodiments of the fused hIL10 polypeptide, PEG is optionally covalently bonded to the N-terminus of hIL10A via a linker, as follows: TIFF2026521391000005.tif17128 is a 40kD branched PEG molecule containing two 20kD arms. In certain embodiments, the PEG is covalently bonded to the N-terminus of hIL10A via an aldehyde linker.

[0033] In some embodiments, the fused hIL10 polypeptide exhibits a proportion of wild-type hIL10 activity in activated human monocytes that is greater than the proportion of wild-type hIL10 activity in activated human CD8 T cells. In some embodiments, the wild-type hIL10 activity is the induction of intracellular STAT3 signaling. In some embodiments, the fused hIL10 polypeptide exhibits an Emax of at least 30%, optionally at least 40%, and optionally at least 50% of the level of wild-type hIL10 activity in activated human monocytes, and the wild-type hIL10 activity in activated human monocytes is selected from the group consisting of inhibition of IL1β secretion and inhibition of TNFα secretion. In some embodiments, the fused hIL10 polypeptide exhibits an Emax of less than 30%, optionally less than 20%, and optionally less than 10% of the level of wild-type hIL10 activity in activated human CD8 T cells, and the wild-type hIL10 activity in activated human CD8 T cells is selected from the group consisting of IFNγ secretion, granzyme A secretion, and granzyme B secretion.

[0034] In other aspects, this disclosure features nucleic acid sequences encoding the fusion hIL10 polypeptide described herein. In some embodiments, the nucleic acid sequence is mRNA. In some embodiments, the nucleic acid sequence is DNA.

[0035] In another aspect, this disclosure provides a vector comprising a nucleic acid sequence described herein that is functionally linked to an expression regulatory sequence. In some embodiments, the vector is a viral vector.

[0036] In another aspect, this disclosure provides cells transformed by the vectors described herein. In some embodiments, the cells are mammalian cells.

[0037] In other contexts, this disclosure is: The active ingredient is (a) a fusion hIL10 polypeptide as described herein, (b) a nucleic acid sequence encoding the fusion hIL10 polypeptide as described herein, (c) a vector comprising the nucleic acid sequence, or (d) cells transformed by a vector comprising the nucleic acid sequence. One or more pharmaceutically acceptable solvents, carriers, stabilizers, preservatives or diluents We provide pharmaceutical preparations that include [the specified ingredient].

[0038] In another context, this disclosure relates to a method for preventing or treating a mammalian subject suffering from a disease, disorder or condition, (a) a fusion hIL10 polypeptide as described herein, (b) a nucleic acid sequence encoding a fusion hIL10 polypeptide as described herein, (c) a vector comprising the nucleic acid sequence, or (d) cells transformed by the vector comprising the nucleic acid sequence, (e) Pharmaceutical preparations described herein The present invention provides a method that includes the step of administering a substance to a target.

[0039] In some embodiments, the disease, disorder, or condition is an autoimmune disease, disorder, or condition. In certain embodiments, the autoimmune disease, disorder, or condition is inflammatory bowel disease (IBD). In certain embodiments, IBD is Crohn's disease or ulcerative colitis.

[0040] In some embodiments, a disease, disorder, or condition is cancer. In certain embodiments, cancer is cancer resulting from chronic inflammation.

[0041] In some aspects, the disease, disorder, or condition is macrophage activation syndrome.

[0042] In some aspects, the disease, disorder, or condition is interferon-gamma induced anemia.

[0043] In another context, this disclosure relates to formula (hIL10A)-L nA method for producing a fused human IL10 (hIL10) polypeptide of -(hIL10B) is provided, comprising the steps of: transfecting host cells with a vector containing a nucleic acid sequence encoding the fused IL10 polypeptide described herein; culturing the cells in a culture medium under conditions that enable expression of the nucleic acid sequence encoding the fused hIL10 polypeptide; and isolating the fused hIL10 polypeptide from the culture medium, optionally further comprising the step of purifying the fused hIL10 polypeptide.

[0044] In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is an E. coli cell.

[0045] In some embodiments, hIL10A is a polypeptide selected from the group consisting of SEQ ID NO: 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, and hIL10B is a polypeptide selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, and 156. In a particular embodiment, hIL10A is a polypeptide selected from the group consisting of SEQ ID NO: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, and 186. [Brief explanation of the drawing]

[0046] [Figure 1A] Figure 1A shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated monocytes of the indicated fused hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the examples. [Figure 1B]Figure 1B shows the results of assays to evaluate the level of STAT3 production (y axis) in activated CD8 T cells in response to the fused hIL10 polypeptide shown, in response to various concentrations (x axis), as described in more detail in the examples. [Figure 2] Figure 2A shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated monocytes with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the examples. Figure 2B shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated CD8 T cells with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the examples. [Figure 3] Figure 3A shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated monocytes with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the Examples. Figure 3B shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated CD8 T cells with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the Examples. [Figure 4] Figure 4A shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated monocytes with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the Examples. Figure 4B shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated CD8 T cells with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the Examples. [Figure 5]Figure 5A shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated monocytes with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the Examples. Figure 5B shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated CD8 T cells with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the Examples. [Figure 6] Figure 6A shows the results of an assay to evaluate the secretion of IL1β (y axis) from activated monocytes in response to fused hIL10 polypeptides at various concentrations (x axis), as described in more detail in the Examples. Figure 6B shows the results of an assay to evaluate the secretion of TNFα (y axis) from activated monocytes in response to fused hIL10 polypeptides at various concentrations (x axis), as described in more detail in the Examples. [Figure 7A] Figure 7A shows the results of an assay to evaluate IFNγ (y axis) production in activated CD8+ T cells in response to various concentrations (x axis) of fused hIL10 polypeptides, as described in more detail in the examples. [Figure 7B] Figure 7B shows the results of an assay to evaluate granzyme A (y axis) production in activated CD8+ T cells in response to various concentrations (x axis) of the fused hIL10 polypeptide, as described in more detail in the examples. [Figure 7C] Figure 7C shows the results of an assay to evaluate the production of granzyme B (y-axis) in activated CD8+ T cells in response to fused hIL10 polypeptides at various concentrations (x-axis), as described in more detail in the examples. As illustrated in Figures 7A to 7C, fused hIL10 polypeptides containing the T100L mutation showed reduced induction of IFNγ, granzyme A, and granzyme B in CD8 T cell blasts compared with fused hIL10 polypeptides, and showed a decrease in Emax compared with fused WT hIL10 polypeptides when evaluated up to a concentration of 100 nM. [Figure 8] Figure 8A shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated monocytes with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the Examples. Figure 8B shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated CD8 T cells with the indicated fusion hIL10 polypeptide in response to various concentrations (x axis), as described in more detail in the Examples. [Figure 9] Figure 9A shows the results of an assay to evaluate the secretion of IL1β (y axis) from LPS-activated monocytes in response to fused hIL10 polypeptides at various concentrations (x axis), as described in more detail in the examples. Figure 9B shows the results of an assay to evaluate the secretion of TNFα (y axis) from LPS-activated monocytes in response to fused hIL10 polypeptides at various concentrations (x axis), as described in more detail in the examples. [Figure 10A] Figure 10A shows the results of an assay to evaluate IFNγ (y axis) production in activated CD8+ T cells in response to various concentrations (x axis) of the fused hIL10 polypeptide, as described in more detail in the examples. [Figure 10B] Figure 10B shows the results of an assay to evaluate granzyme A (y axis) production in activated CD8+ T cells in response to various concentrations (x axis) of the fused hIL10 polypeptide, as described in more detail in the examples. [Figure 10C] Figure 10C shows the results of an assay to evaluate granzyme B (y axis) production in activated CD8+ T cells in response to various concentrations (x axis) of the fused hIL10 polypeptide, as described in more detail in the examples. [Figure 11]Figure 11A shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated mouse bone marrow cells of the indicated fusion mRNA in response to various concentrations (x axis), as described in more detail in the Examples. Figure 11B shows the results of an assay to evaluate the level of STAT3 production (y axis) in activated mouse CD8 T cells of the indicated fusion mRNA in response to various concentrations (x axis), as described in more detail in the Examples. [Figure 12] Figure 12A shows the results of an assay to evaluate IFNγ (y axis) production in LPS-activated mouse splenocytes in response to various concentrations (x axis) of fused mRNA polypeptide, as described in more detail in the Examples. Figure 12B shows the results of an assay to evaluate IL6 (y axis) production in LPS-activated mouse splenocytes in response to various concentrations (x axis) of fused mRNA polypeptide, as described in more detail in the Examples. [Figure 13] Figure 13 shows the results of an assay to evaluate the level of granzyme B secretion (y axis) in activated mouse CD8 T cells in response to the fused mRNA in various concentrations (x axis), as described in more detail in the examples. [Figure 14] Figure 14 shows the results of an assay to evaluate viable (%) activated mouse CD8 T cells (y axis) in response to various concentrations of fused mRNA (x axis), as described in more detail in the examples. As illustrated, the mouse fusion polypeptide with the T100L mutation was weak in inducing CD8 T cell blast survival and did not achieve Emax levels equivalent to those of the fused mRNA (WT) polypeptide up to a concentration of 100 nM. [Figure 15]Figure 15 shows the results of an assay to evaluate the induction of cell surface CD64 (y axis) in mouse monocytes in response to various concentrations of the fusion mRNA (x axis), as described in more detail in the examples. The provided data show that the fusion polypeptide with the T100L mutation (TP0015) showed reduced induction of CD64 cell surface expression on monocytes compared to the native mRNA molecule. [Figure 16] Figure 16 provides the results of a pharmacokinetic study to evaluate the effect of serum concentration in mice over time (y-axis) in response to a single dose of various indicated doses of the indicated PEGylated mouse fusion mIL10 polypeptide containing the T100L mutation, as described in more detail in the Examples. The provided data show that the PEGylated version of the fusion mIL10 polypeptide exhibits a longer in vivo half-life than the non-PEGylated fusion mIL10 polypeptide after a single dose of the polypeptide. [Figure 17] Figure 17 provides the results of an analytical SEC monitoring the pH-dependent stability of natural IL10 (hIL10 WT) or fused IL10 WT (h_SS0052_AA). The provided data show that the natural IL10 molecule dissociated into monomers at pH < 5.0, while the fused hIL10 molecule remained stable under similar conditions. [Figure 18]Figures 18A to 18D provide schematic diagrams of various configurations of fused IL10 polypeptides conjugated to Fc polypeptides. Figures 18A to C show the fused IL10 polypeptide conjugated to the "hole" subunit of the KiH dimer Fc(18A), the fused IL10 polypeptide conjugated to the "knob" subunit of the KiH dimer Fc(18B), the first (white) fused IL10 polypeptide conjugated to the "hole" Fc subunit, and the second (black) fused IL10 polypeptide conjugated to the "knob" Fc subunit of the KiH dimer Fc(18C), showing the first and second fused IL10 polypeptides with different knob-into-hole (KiH)Fc configurations. Figure 18D illustrates a configuration in which Fc is not modified to promote heterodimerization, and each of the Fc subunits of the Fc dimer is conjugated to a fused IL10 polypeptide. [Figure 19] Figures 19A and 19B provide the results of an evaluation of interspecies cross-reactivity of human molecules in mouse splenocytes, showing IL6 levels (A, y-axis) and TNFα levels (B, y-axis) in response to various concentrations of test substances (x-axis), as described in more detail in Example 11 herein. [Figure 20] Figure 20 provides the results of evaluating granzyme B secretion (vertical axis) as a percentage of wild-type IL10 activity in response to various concentrations of the test substance (horizontal axis), as described in more detail in Example 12 herein. [Figure 21] Figure 21 provides the results of evaluating the activity of the test substance, assessed by its ability to inhibit LPS-induced secretion of IL-6 in cynomolgus monkey whole blood in response to various concentrations of the test substance (horizontal axis), as described in more detail in Example 13 herein. [Figure 22] Figure 22 provides the results of evaluating body weight over time (horizontal axis) (vertical axis) in mice treated with various test substances at various doses in a DSS model of ulcerative colitis, as described in more detail in Example 14 herein. [Figure 23]Figure 23 provides the results of evaluating the percentage of initial body weight over time (horizontal axis) in mice treated with various test substances at various doses in a DSS model of ulcerative colitis, as described in more detail in Example 14 herein. [Figure 24] Figure 24 provides the results of evaluating body weight (vertical axis) in response to various test substances (horizontal axis) at various doses at the end of a DSS model of ulcerative colitis, as described in more detail in Example 14 herein. [Figure 25] Figure 25 provides the results of evaluating colon length (vertical axis) in response to various doses of various test substances (horizontal axis) at the end of a DSS model of ulcerative colitis, as described in more detail in Example 14 herein. [Figure 26] Figure 26 provides hematocrit results (vertical axis) obtained from mice on day 4 of a DSS model of ulcerative colitis in response to various tests at various doses (horizontal axis), as described in more detail in Example 14 herein. [Figure 27] Figure 27 provides the results of the evaluation of serum cytokine levels (vertical axis) of IFNγ, IL10, IL-1B, IL6, TNF-α, and IL-4 in response to the administration of various test substance treatments over time (horizontal axis), as described in more detail in Example 14 herein. [Figure 28] Figure 28 provides the results of the evaluation of serum cytokine levels (vertical axis) of IL-5, IP10, IL17A / F, and MIP1a in response to the administration of various test substance treatments over time (horizontal axis), as described in more detail in Example 14 herein. [Figure 29] Figure 29 shows the results of evaluating CD163 expression by peritoneal macrophages (vertical axis) in response to administration of various test substances (horizontal axis), as described in more detail in Example 14. [Figure 30] Figure 30 shows the results of evaluating CD64 expression by peritoneal macrophages (vertical axis) in response to administration of various test substances (horizontal axis), as described in more detail in Example 14. [Figure 31]Figure 31 provides the results of evaluating the percentage of epithelial damage (vertical axis) in the mouse colon in response to the administration of various test substances (horizontal axis), as described in more detail in Example 14. [Figure 32] Figure 32 provides the results of evaluations of CD11B+ cells and TH17 cells (vertical axis) in mouse colonic mucosa in response to the administration of various test substances (horizontal axis), as described in more detail in Example 14. [Figure 33] Figure 33 provides the results of evaluating the level of pSTAT3 induction in peripheral blood immune cells in response to the administration of various test substances (horizontal axis), as described in more detail in Example 15. [Figure 34] Figure 34 provides the results of pharmacokinetic studies to evaluate the serum concentrations of various test substances over time, as described in more detail in Example 16. [Figure 35] Figure 35 provides the results of evaluations of IL6, KC / GRO, IL10, and TNFα serum cytokine levels (vertical axis) at 1.5 hours after LPS administration in an LPS shock model, in response to the administration of various test substances (horizontal axis), as described in more detail in Example 17. [Figure 36] Figure 36 provides the results of evaluations of serum cytokine levels (vertical axis) of IL6, KC / GRO, IL10, TNFα, IFNγ, IL1b, and IL5 at 6 hours after LPS administration in an LPS shock model, in response to the administration of various test substances (horizontal axis), as described in more detail in Example 17. [Figure 37] Figure 37 provides the results of pharmacokinetic studies to evaluate the serum concentrations of various test substances over time, as described in more detail in Example 18. [Figure 38] Figure 38 provides the results of evaluating the ability of the test substance to induce pSTAT3 in peripheral blood myeloid cells over time (horizontal axis), as described in more detail in Example 19 (vertical axis). [Figure 39]Figure 39 provides the results of evaluating the time course of peritoneal exudative immune cell scavenger CD163 and CD64 receptor expression (vertical axis) in response to the administration of various test substances (horizontal axis), as described in more detail in Example 21. [Figure 40] Figure 40 provides the results of evaluating the expression of the peritoneal exudative immune cell scavenger CD16 over time (vertical axis) in response to the administration of various test substances (horizontal axis), as described in more detail in Example 22. [Figure 41] Figure 41 provides the results of evaluating the expression of the peritoneal exudative immune cell scavenger CD38 over time (vertical axis) in response to the administration of various test substances (horizontal axis), as described in more detail in Example 23. [Figure 42] Figure 42 provides the results of evaluating CD38 and CD16 levels (vertical axis) on peripheral blood immune cells over time in response to the administration of various test substances (horizontal axis), as described in more detail in Example 24. [Figure 43] Figure 43 provides the results of evaluating CD150 levels on peripheral blood B cells over time in response to the administration of various test substances, as described in more detail in Example 25. [Figure 44] Figure 44 provides hematocrit results (vertical axis) obtained from mice in an IFNγ-induced anemia model on day 8 of the study, in response to the administration of various test substances, as described in more detail in Example 26. [Figure 45] Figure 45 provides data on the secretion of IL1βb (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 27. [Figure 46] Figure 46 provides data on the secretion of IL-6 from human monocytes stimulated with LPS in response to increasing concentrations of various test substances (horizontal axis), as described in more detail in Example 27. [Figure 47]Figure 47 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 27. [Figure 48] Figure 48 provides data on the secretion of IL1βb from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 28. [Figure 49] Figure 49 provides data on the secretion of TNFα (vertical axis) from LPS-stimulated human monocytes in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 28. [Figure 50] Figure 50 provides data on the secretion of IL1βb from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 29. [Figure 51] Figure 51 provides data on the secretion of TNFα (vertical axis) from LPS-stimulated human monocytes in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 29. [Figure 52] Figure 52 provides data on the secretion of IL1βb from human monocytes stimulated with LPS (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 30. [Figure 53] Figure 53 provides data on the secretion of IL6 from human monocytes stimulated with LPS (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 30. [Figure 54] Figure 54 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 30. [Figure 55]Figure 55 provides data on the secretion of IL1βb from human monocytes stimulated with LPS (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 31. [Figure 56] Figure 56 provides data on the secretion of IL-6 from human monocytes stimulated with LPS in response to increasing concentrations of various test substances (horizontal axis), as described in more detail in Example 31. [Figure 57] Figure 57 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 31. [Figure 58] Figure 58 provides data on the secretion of IL1βb (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 32. [Figure 59] Figure 59 provides data on the secretion of IL-6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 32. [Figure 60] Figure 60 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 32. [Figure 61] Figure 61 provides data on the secretion of IL1βb (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 33. [Figure 62] Figure 62 provides data on the secretion of IL6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 33. [Figure 63]Figure 63 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 33. [Figure 64] Figure 64 provides data on the secretion of IL1βb from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 34. [Figure 65] Figure 65 provides data on the secretion of IL6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 34. [Figure 66] Figure 66 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 34. [Figure 67] Figure 67 provides data on the secretion of IL1βb from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 35. [Figure 68] Figure 68 provides data on the secretion of IL6 from human monocytes stimulated with LPS (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 35. [Figure 69] Figure 69 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 35. [Figure 70] Figure 70 provides data on the secretion of IL1βb from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 36. [Figure 71]Figure 71 provides data on the secretion of IL6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 36. [Figure 72] Figure 72 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 36. [Figure 73] Figure 73 provides data on the secretion of IL1βb from human monocytes stimulated with LPS (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 37. [Figure 74] Figure 74 provides data on the secretion of IL-6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 37. [Figure 75] Figure 75 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 37. [Figure 76] Figure 76 provides data on the secretion of IL1βb from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 38. [Figure 77] Figure 77 provides data on the secretion of IL6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 38. [Figure 78] Figure 78 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 38. [Figure 79]Figure 79 provides data on the secretion of IL1βb from human monocytes stimulated with LPS (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 39. [Figure 80] Figure 80 provides data on the secretion of IL6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 39. [Figure 81] Figure 81 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 39. [Figure 82] Figure 82 provides data on the secretion of IL1βb from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 40. [Figure 83] Figure 83 provides data on the secretion of IL6 from human monocytes stimulated with LPS in response to increasing concentrations of various test substances (horizontal axis), as described in more detail in Example 40. [Figure 84] Figure 84 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 40. [Figure 85] Figure 85 provides data on the secretion of IL1βb from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 41. [Figure 86] Figure 86 provides data on the secretion of IL-6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 41. [Figure 87]Figure 87 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 41. [Figure 88] Figure 88 provides data on the secretion of IL1βb (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 42. [Figure 89] Figure 89 provides data on the secretion of IL6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 42. [Figure 90] Figure 90 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 42. [Figure 91] Figure 91 provides data on the secretion of IL1βb from human monocytes stimulated with LPS (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 43. [Figure 92] Figure 92 provides data on the secretion of IL6 from human monocytes stimulated with LPS (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 43. [Figure 93] Figure 93 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 43. [Figure 94] Figure 94 provides data on the secretion of IL1βb (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 44. [Figure 95]Figure 95 provides data on the secretion of IL6 from LPS-stimulated human monocytes (vertical axis) in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 44. [Figure 96] Figure 96 provides data on the secretion of TNFα (vertical axis) from human monocytes stimulated with LPS in response to increasing concentrations (horizontal axis) of various test substances, as described in more detail in Example 44. [Figure 97] Figure 97 provides data on the IC50 values ​​(vertical axis) of various test molecules when inhibiting IL1βb from human monocytes stimulated with LPS, as described in more detail in Example 45. [Figure 98] Figure 98 provides data on the IC50 values ​​(vertical axis) of various test molecules when inhibiting IL6 from human monocytes stimulated with LPS, as described in more detail in Example 45. [Figure 99] Figure 99 provides data on the IC50 values ​​(vertical axis) of various test molecules when inhibiting TNFα from human monocytes stimulated with LPS, as described in more detail in Example 45. [Figure 100] Figure 100 provides data on the secretion of granzyme A of various test products, normalized to the percentage maximum secretion induced by fusion_WT IL-10(h_DR2339_AA), as described in more detail in Example 46. [Figure 101] Figure 101 provides data on the secretion of granzyme B of various test products, normalized to the percentage maximum secretion induced by fusion_WT IL-10(h_DR2339_AA), as described in more detail in Example 46. [Figure 102] Figure 102 provides data on IFNg secretion of various test substances normalized to the percentage maximal secretion induced by fusion_WT IL-10(h_DR2339_AA), as described in more detail in Example 46. [Figure 103]Figure 103 provides data on IFNg secretion (vertical axis) in human CD8 T cell blasts from two human donors, normalized to the percentage maximal secretion induced by fused h_WT IL-10 (h_DR2339_AA), in response to various concentrations of the test substance (horizontal axis), as described in further detail in Example 47. Each panel in Figure 103 represents data from cells obtained from different human donors, upper panel donor RG3708 and lower panel donor RG2889. [Figure 104] Figure 104 provides data on the secretion of granzyme A in human CD8 T cell blasts, normalized to the percentage maximal secretion induced by fused h_WT IL-10 (h_DR2339_AA), in response to various concentrations of the test substance (horizontal axis), as described in more detail in Example 48 (vertical axis). [Figure 105] Figure 105 provides data on the secretion of granzyme B in human CD8 T cell blasts, normalized to the percentage maximal secretion induced by fused h_WT IL-10 (h_DR2339_AA), in response to various concentrations of the test substance (horizontal axis), as described in more detail in Example 48 (vertical axis). [Figure 106] Figure 106 provides data on IFNg secretion (vertical axis) in human CD8 T cell blasts, normalized to the percentage maximal secretion induced by fused h_WT IL-10 (h_DR2339_AA), in response to various concentrations of the test substance (horizontal axis), as described in more detail in Example 48. [Modes for carrying out the invention]

[0047] Detailed explanation of disclosure 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Throughout this disclosure, amino acids will be referenced by one-letter or three-letter codes.

[0055] 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).

[0056] 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 a native amino acid found in the wild-type molecule. In this disclosure, the numbering of amino acid residues in human IL10 polypeptide monomers is done by referring to the number of residues in the “mature” form of the hIL10 polypeptide monomer provided in SEQ ID NO:4. With respect to human IL10 polypeptide monomers, substitutions are indicated herein by a one-letter amino acid code followed by the wild-type hIL10 (SEQ ID NO:4) amino acid position followed by the one-letter amino acid code being substituted. For example, the human IL10 polypeptide monomer having the modification “D25K” means that the aspartic acid (D) residue at position 25 of (SEQ ID NO:4) 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.

[0057] 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.

[0058] definition Unless otherwise specified, the following terms are intended to have the meanings set forth below. Other terms are defined elsewhere throughout this specification.

[0059] 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.

[0060] 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.

[0061] 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 binding of a molecule 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, and its ability to modulate immunological activities such as gene expression, cell proliferation, and inflammatory responses. “Activity” is typically expressed as the level of biological activity per unit of the active agent being tested, 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 activity that promotes cell proliferation and replication, including dysregulated cell division as observed in neoplasms, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis, and angiogenesis.

[0062] Administer / AdministrationThe terms “administer” and “administer” are used herein without distinction to refer to any act of contacting a subject, including, in vitro, in vivo, or ex vivo, cells, tissues, organs, or bodily fluids of the subject with an active substance (e.g., hIL10 mutein, or engineered cells expressing hIL10 mutein, chemotherapeutic agents, antibodies, or a pharmaceutical formulation comprising one or more of the foregoing). The administration of the active substance may be achieved by any of the various methods recognized in the art, without limitation, including local 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.

[0063] 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 )

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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%.

[0068] 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). For example, each of the following groups of amino acids may be considered conserved amino acids: (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.

[0069] ~ 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 mutaine, the reference sequence may be the amino acid sequence of wild-type hIL10 (SEQ ID NO: 4).

[0070] ~ 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: 4)) or a polynucleotide sequence (e.g., the cDNA encoding wild-type 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.

[0071] Effective concentration (EC): As used herein, the term "effective concentration" or its abbreviation "EC" is used interchangeably to refer to the concentration of an agent sufficient to effect 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 a test agent. When the magnitude of the response is expressed as a function of the concentration ("C") of the test agent, the abbreviation "EC" is used. In the context of a biological system, the term Emax refers to the maximum magnitude of a given biological effect observed in response to a saturating concentration of an activating test agent. When a subscript (e.g., EC 40 , EC 50 , etc.) is provided to the abbreviation EC, the subscript refers to the percentage of Emax of the biological response observed at that concentration. For example, in a test system, the concentration of a test agent sufficient to effect an induction of a measurable biological parameter that is 30% of the maximum level of such a measurable biological parameter in response to such a test agent is referred to as the "EC 30 " of the test agent with respect to such a biological parameter. Similarly, the term "EC 100 " is used to denote the effective concentration of an agent that results in a maximal (100%) response of a measurable parameter in response to such an agent. Similarly, the term EC 50 (commonly used in the field of pharmacodynamics) refers to the concentration of an agent sufficient to effect a change that is half (about 50%) of the maximum value of a measurable parameter. The term "saturating concentration" refers to the maximum possible amount of a test agent that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacodynamics, the saturating concentration of a drug is typically used to denote a sufficient concentration of the drug such that all available receptors are occupied by the drug, and EC 50 is the drug concentration that gives an effect that is half of the maximum value.

[0072] 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).

[0073] 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.

[0074] 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 having a distinct additional (e.g., complementary, particularly 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. In some embodiments, fusion proteins also include the IL10 fusion protein homodimer of this disclosure.

[0075] 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).

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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-adjacent intracellular domains.

[0081] 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.

[0082] 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).

[0083] 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.

[0084] 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 hIL10 mutein monomer can activate IL10 signaling in cells expressing the IL10 receptor as part of a homodimer, but may have improved properties compared to an unmodified polypeptide. The term “modified” includes amino acid substitutions not present in parent IL10 or wild-type IL10, and includes variants and muteins of IL10 polypeptides.

[0085] 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) and antagonists.

[0086] Mutain As used herein, the term “mutein” is used to refer to a variant of a wild-type polypeptide, which includes modifications to the primary structure (i.e., amino acid sequence) of such polypeptide. A mutein may have at least 99% sequence identity with respect to the parent polypeptide from which it is derived, or at least 98% sequence identity, or at least about 97% sequence identity, or at least 96% sequence identity, or at least 95% sequence identity, or at least about 94% sequence identity, or at least 93% sequence identity, or at least 92% sequence identity, or at least 91% sequence identity, or at least 90% sequence identity.

[0087] nucleic acidAs 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.

[0088] One or more amino acid substitutions As 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 the reference sequence. In some embodiments, the reference sequence is the wild-type hIL10 monomer (SEQ ID NO: 4).

[0089] 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.

[0090] 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.”

[0091] 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%.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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)).

[0096] 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.

[0097] 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)).

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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."

[0103] 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.

[0104] subjectThe terms “recipient,” “individual,” “subject,” and “patient” are used herein without distinction and refer to any mammalian subject, in particular human, for whom 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 embodiments, mammal is human.

[0105] 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.

[0106] ~ 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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 hIL10 variant polypeptide monomer 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 in 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.

[0111] 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 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.

[0112] 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.

[0113] 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.

[0114] Human IL-10: Human IL-10 (hIL-10) is a non-covalently linked homodimer protein containing two identical subunits. Each hIL-10 monomer is expressed as a 178-amino acid preprotein containing an 18-amino acid signal sequence (SEQ ID NO: 5), which is removed post-translation to form a 160-amino acid mature protein. The canonical amino acid sequence of the mature ("wild-type") IL-10 protein (UniProt reference number P22301) without the signal sequence (corresponding to amino acids 19-178 of the preprotein) is as follows: TIFF2026521391000006.tif17145

[0115] Fusion Human IL-10 Polypeptide This disclosure is based on the following formula: (hIL10A)-L n -(hIL10B) The following fusion human IL10 (hIL10) polypeptides are provided, where hIL10A and hIL10B are independently selected from the group consisting of wild-type hIL10 (SEQ ID NO: 4) and hIL10 mutein, at least one of hIL10A or hIL10B is hIL10 mutein, L is a linker, and n=0 (non-existent) or 1 (existent).

[0116] hIL10 Mutein In some embodiments, formula (hIL10A)-L n In the fusion hIL10 polypeptide -(hIL10B), at least one of hIL10A and hIL10B is hIL10 mutain. In some cases, hIL10 mutain may contain one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96 and R104 of SEQ ID NO:4. In some embodiments, hIL10 mutein comprises a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with respect to SEQ ID NO:4, and includes one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96, and R104 of SEQ ID NO:4. In some cases, hIL10 mutein may include one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96, and R104 of SEQ ID NO:4. In some embodiments, the hIL10 mutein comprises a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with respect to SEQ ID NO:4, and comprises one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96, and R104 of SEQ ID NO:4.

[0117] In some embodiments, hIL10 mutein includes an amino acid substitution at position T100, for example, T100D, T100V, T100E, T100A, T100R, T100N, T100Q, T100E, T100I, T100L, T100K, T100M, or T100S.

[0118] In some embodiments, the hIL10 mutant comprises an amino acid substitution at position H14, such as H14C, H14F, H14P, H14W, H14G, H14A, H14D, H14E, H14I, H14K, H14L, H14M, H14N, H14Q, H14R, H14S, H14T, H14Y or H14V.

[0119] In some embodiments, the hIL10 mutant comprises an amino acid substitution at position N18, such as N18Y, N18F, N18A, N18D, N18E, N18L, N18V, N18S, N18T, N18I, N18V, N18M, N18R and N18K, or N18H.

[0120] In some embodiments, the hIL10 mutant comprises an amino acid substitution at position N21, such as N21A, N21R, N21Q, N21H, N21K, N21S, N21V, N21I, N21L, N21M, N21T N21C, N21D or N21E.

[0121] In some embodiments, the hIL10 mutant comprises an amino acid substitution at position M22, such as M22A, M22V, M22I, M22L, M22N, M22D, M22S, M22T, M22W or M22Q.

[0122] In some embodiments, the hIL10 mutant comprises an amino acid substitution at position R24, such as R24E, R24D, R24N, R24Q, R24A, R24S or R24T.

[0123] In some embodiments, the hIL10 mutant comprises an amino acid substitution at position D25, such as D25A, D25N, D25H, D25I, D25K, D25L, D25P, D25Q or D25V.

[0124] In some embodiments, the hIL10 mutant comprises an amino acid substitution at position D28, such as D28A, D28E, D28L, D28V, D28S, D28T, D28I, D28V, D28M, D28H, D28K or D28R.

[0125] In some embodiments, hIL10 mutaine includes an amino acid substitution at position R32, for example, R32A, R32D, R32E, R32L, R32V, R32S, R32T, R32I, R32V, R32M, R32N, R32Q, R32G, R32C, R32P, R32F, R32Y, or R32H.

[0126] In some embodiments, hIL10 mutein includes an amino acid substitution at position E74, for example, E74A, E74D, E74L, E74V, E74S, E74T, E74I, E74V, E74M, E74H, E74K, or E74R.

[0127] In some embodiments, hIL10 mutein includes an amino acid substitution at position H90, for example, H90A, H90D, H90E, H90I, H90K, H90L, H90M, H90N, H90Q, H90R, H90S, H90T, H90Y, or H90V.

[0128] In some embodiments, hIL10 mutaine includes an amino acid substitution at position N92, for example, N92D, N92Q, N92E, N92H, N92K, N92S, N92V, N92I, N92L, N92M, N92T, or N92A.

[0129] In some embodiments, hIL10 mutein includes an amino acid substitution at position S93, for example, S93E, S93A, S93R, S93N, S93D, S93Q, S93E, S93I, S93L, S93K, S93M, S93G, or S93V.

[0130] In some embodiments, hIL10 mutein includes an amino acid substitution at position E96, for example, E96C, E96F, E96Y, E96W, E96A, E96N, E96D, E96Q, E96H, E96K, or E96S.

[0131] In some embodiments, hIL10 mutein includes an amino acid substitution at position R104, for example, R104A, R104W, R104Y, R104F, R104H, R104D, R104E, R104N, R104Q, R104S, R104T, R104I, R104L, R104V, or R104M.

[0132] Formula (hIL10A)-L n Examples of hIL10 mutaines that can be used in the fused hIL10 polypeptide of -(hIL10B) are provided in Table 1A below.

[0133] (Table 1A) TIFF2026521391000007.tif210147TIFF2026521391000008.tif210147TIFF2026521391000009.tif98147

[0134] Formula (hIL10A)-L n Table 1B below provides examples of hIL10 mutaines that can be used in the fused hIL10 polypeptide of -(hIL10B).

[0135] (Table 1B) TIFF2026521391000010.tif75147TIFF2026521391000011.tif210147TIFF2026521391000012.tif75147

[0136] Optionally, in some embodiments, the hIL10 mutein may further include the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues at the N-terminus of the mutein, in addition to one or more amino acid substitutions compared to SEQ ID NO:4. In certain embodiments, hIL10A in the fusion hIL10 polypeptide has the amino-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to SEQ ID NO:4. In other embodiments, hIL10B in the fusion hIL10 polypeptide has the amino-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to SEQ ID NO:4. In certain embodiments, hIL10A in the fusion hIL10 polypeptide has the amino-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to the amino acid sequence in Table 1A. In other embodiments, hIL10B in the fused hIL10 polypeptide has amino-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to the amino acid sequence in Table 1A. In some embodiments, hIL10A is selected from the amino acid sequence in Table 1B, and hIL10B is selected from the amino acid sequence in Table 1A. In some embodiments, the present disclosure is (hIL10A)-L n The formula -(hIL10B) provides a fused hIL10 polypeptide, where hIL10A is a polypeptide selected from the group consisting of SEQ ID NO: 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 and 205, and hIL10B is a polypeptide selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155 and 156.

[0137] Linker In some embodiments, (hIL10A)-L n In the equation -(hIL10B), hIL10A and hIL10B should be linked or fused together by linker L, i.e., n is 1 in the equation.

[0138] In some embodiments, the linker is a polypeptide with 1 amino acid (e.g., Thr) to 50 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-40, 40-50, 10-30, 20-40, or 30-50 amino acids) or more than 50 amino acids.

[0139] Examples of linkers include, without limitation, (G) x (A) x , (S) x , (GA) x , (GS) x , (AS) x , (AG) x , (SG) x , (SA) x Examples include, where x can be an integer, for example, an integer between 1 and 50. Examples of linkers containing glycine and serine include, but are not limited to, (GmSo) z (SEQ ID NO:129), (GSGGS) z (SEQ ID NO:130), (GmSoGm) z (SEQ ID NO:131), (GmSoGmSoGm) z (SEQ ID NO:132), (GSGGSm) z (SEQ ID NO:133), (GSGSmG) z (SEQ ID NO:134) and (GGGSm) z Examples include (SEQ ID NO:135), and combinations thereof (wherein m, z, and o are independently selected from at least 1 to 20 integers (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, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)) and other flexible linkers. In some embodiments, the polypeptide linker has a structure (GGGGS m ) n (SEQ ID NO:136), (GGGS) m ) n (SEQ ID NO:137), (GGGAm ) n (SEQ ID NO:138) and (GGGGA m ) n (SEQ ID NO:139), and glycine - serine polymers of combinations thereof, wherein m, n, and o are each independently selected from 1, 2, 3, or 4. Exemplary glycine - serine linkers include, without limitation, monomer: GGGGS (referred to as "G4S"; SEQ ID NO:104), GGGGA (referred to as "G4A"; SEQ ID NO:112), GGGS (referred to as "G3S"; SEQ ID NO:103)), and GGGA (referred to as "G3A" (SEQ ID NO:140)), or their homopolymers (e.g., "GGGGSGGGGS" also referred to as (G4S)2; SEQ ID NO:111) or heteropolymers (e.g., GGGGSGGGS also referred to as G4S - G3S; SEQ ID NO:108). Specific linker sequences useful in the practice of the present disclosure include, without limitation, the following: TIFF2026521391000013.tif51145 is included. In the construction of such polymers, it may be desirable to avoid repetitive "GSG" sequences that can potentially introduce non - naturally occurring glycosylation sites.

[0140] In some embodiments, the polypeptide linker of the fusion hIL10 polypeptide of the formula (hIL10A)-L n -(hIL10B) has a sequence selected from GGGSGSGSGSG (SEQ ID NO:19) or NQMFDQKYDDP (SEQ ID NO:20). In some embodiments, the linker in the fusion hIL10 polypeptide of the formula (hIL10A)-L n -(hIL10B) is Thr.

[0141] In some embodiments, the linker in the fusion hIL10 polypeptide of the formula (hIL10A)-L n -(hIL10B) can be a single amino acid. For example, in some embodiments, the single amino acid is T, S, A, or G (e.g., T).

[0142] Fusion hIL10 polypeptide In some embodiments, formula (hIL10A)-L n The fusion hIL10 polypeptide of -(hIL10B) can contain two identical IL10 monomers, i.e., hIL10A and hIL10B in the formula are both wild-type IL10 molecules (e.g., SEQ ID NO:4), or both are the same hIL10 mutain. In some embodiments, the formula (hIL10A)-L n The fusion hIL10 polypeptide -(hIL10B) can contain two different IL10 monomers, i.e., one of hIL10A and hIL10B is a wild-type IL10 molecule (e.g., SEQ ID NO:4) and the other is an hIL10 mutain, or both are different hIL10 mutains.

[0143] In some embodiments, hIL10A and hIL10B are directly fused with each other, i.e., (hIL10A)-L n In the formula -(hIL10B), n is 0 (no linker). hIL10A and hIL10B can be fused with each other via their ends, i.e., the N-terminus of hIL10B is fused with the C-terminus of hIL10A. In some embodiments, hIL10A and hIL10B are fused with each other via a linker, i.e., n is 1. Examples of linkers are described herein.

[0144] In some embodiments, (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which comprises at least one amino acid substitution and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and hIL10B, which comprises at least one amino acid substitution and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4. In some embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions compared to the sequence of SEQ ID NO:4, and hIL10B, which contains a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions compared to the sequence of SEQ ID NO:4.

[0145] In some embodiments, (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) includes hIL10A, which contains one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions at one or more positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96, and R104 of SEQ ID NO:4, and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, as well as SEQ ID hIL10B may include one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions at one or more positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96, and R104 of NO:4, and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4. In some embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) includes hIL10A, which contains one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions at one or more positions corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93, and E96 of SEQ ID NO:4, and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and hIL10A, which contains one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions at one or more positions corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93, and E96 of SEQ ID NO:4, and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and hIL10B may contain a sequence that has at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of NO:4.

[0146] In some embodiments, (hIL10A)-Ln A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains the sequence of the wild-type IL10 molecule (e.g., SEQ ID NO:4), and hIL10B, which contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) identity with respect to the sequence of SEQ ID NO:4.

[0147] In some embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and hIL10B, which contains the sequence of the wild-type IL10 molecule (e.g., SEQ ID NO:4).

[0148] In some embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains a sequence having amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96, and R104 of SEQ ID NO:4, and hIL10B, which contains a sequence having amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96, and R104 of SEQ ID NO:4. In some embodiments, (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains a sequence having amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93, or E96 of SEQ ID NO:4, and hIL10B, which contains a sequence having amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93, and E96 of SEQ ID NO:4.

[0149] In some embodiments, (hIL10A)-L n In the fusion hIL10 polypeptide having the formula -(hIL10B), one or both of hIL10A and hIL10B may have N-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to the sequence of SEQ ID NO:4. In certain embodiments, hIL10A may have N-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to the sequence of SEQ ID NO:4. In certain embodiments, hIL10B may have N-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to the sequence of SEQ ID NO:4.

[0150] In some embodiments, (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) identity with respect to the sequence of SEQ ID NO:4, and hIL10B, which contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions and a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) identity with respect to the sequence of SEQ ID NO:4, and one or both of hIL10A and hIL10B may contain SEQ ID Compared to the sequence of NO:4, it has an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[0151] In some embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains a sequence having amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96, and R104 of SEQ ID NO:4, and hIL10B, which contains a sequence having amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96, and R104 of SEQ ID NO:4, and one or both of hIL10A and hIL10B have N-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to the sequence of SEQ ID NO:4. In some embodiments, (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains a sequence having amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93, or E96 of SEQ ID NO:4, and hIL10B, which contains a sequence having amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93, or E96 of SEQ ID NO:4, wherein one or both of hIL10A and hIL10B have N-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to the sequence of SEQ ID NO:4.

[0152] In some embodiments, this disclosure relates to formula (hIL10A)-L n The provided fusion hIL10 polypeptide -(hIL10B) is an hIL10 mutain selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, wherein one or both of hIL10A and hIL10B have an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to the sequence of SEQ ID NO: 4.

[0153] In some embodiments, this disclosure relates to formula (hIL10A)-L nThe fusion hIL10 polypeptide -(hIL10B) is provided, where hIL10A and hIL10B are hIL10 mutains independently selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 and 205, and optionally one or both of hIL10A and hIL10B have an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues compared to the sequence of SEQ ID NO: 4.

[0154] In some embodiments, this disclosure relates to formula (hIL10A)-L n The fusion hIL10 polypeptide -(hIL10B) is provided, and both hIL10A and hIL10B are hIL10 mutains selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 and 205, and optionally one or both of hIL10A and hIL10B have an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues compared to the sequence of SEQ ID NO: 4.

[0155] In some embodiments, this disclosure relates to formula (hIL10A)-L n-(hIL10B) provides a fusion hIL10 polypeptide, hIL10A containing an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the deletion of the N-terminal amino acid (e.g., the first three or six amino acids of the N-terminus of the fusion IL10 molecule are deleted compared to the wild-type sequence) results in the N-terminal amino acid being glutamine (Q). The N-terminal glutamine residue has been observed to spontaneously cyclize under physiological or near-physiological conditions to form pyroglutamic acid (pE). (See, e.g., Liu, et al (2011) J. Biol. Chem. 286(13):11211-11217). In some embodiments, pyroglutamic acid formation complicates N-terminal PEG conjugation, particularly when aldehyde chemistry is used for N-terminal PEGylation. As a result, when a fusion IL10 molecule containing an N-terminal glutamine is PEGylated, for example, by the deletion of 3 or 6 N-terminal residues from the wild-type sequence, the N-terminal glutamine is replaced by an alternative amino acid. In some embodiments, the N-terminal glutamine residue is selected from the group consisting of E and D.

[0156] In some embodiments, this disclosure relates to formula (hIL10A)-L n -(hIL10B) provides a fusion hIL10 polypeptide, where hIL10A comprises a 3-amino acid N-terminal deletion and an amino acid substitution selected from the group consisting of Q4E and Q4D. In some embodiments, the disclosure provides a fusion of formula (hIL10A)-L n -(hIL10B) provides a fusion hIL10 polypeptide, where hIL10A includes a six-amino acid N-terminal deletion and an amino acid substitution selected from the group consisting of Q7E and Q7D.

[0157] In certain embodiments, (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains an amino acid substitution D25K and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and hIL10B, which contains an amino acid substitution D25K and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and one or both of hIL10A and hIL10B may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4 (e.g., hIL10B may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4. NO:21) has an N-terminal deletion.

[0158] In certain embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains an amino acid substitution N21K and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and hIL10B, which contains an amino acid substitution N21K and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and one or both of hIL10A and hIL10B have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4 (e.g., hIL10B has an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4. NO:21) has an N-terminal deletion.

[0159] In certain embodiments, (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) of the amino acid substitution M22A compared to the sequence of SEQ ID NO:4, and hIL10B, which contains a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) of the amino acid substitution M22A compared to the sequence of SEQ ID NO:4, and one or both of hIL10A and hIL10B may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4 (e.g., hIL10B may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4). NO:21) has an N-terminal deletion.

[0160] In certain embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains an amino acid substitution M22S and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and hIL10B, which contains an amino acid substitution M22S and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and one or both of hIL10A and hIL10B may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4 (e.g., hIL10B may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4. NO:21) has an N-terminal deletion.

[0161] In certain embodiments, (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) of the amino acid substitution T100L compared to the sequence of SEQ ID NO:4, and hIL10B, which contains a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) of the amino acid substitution T100L compared to the sequence of SEQ ID NO:4, and either hIL10A or hIL10B (e.g., hIL10A only or hIL10B only) or both may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4 (e.g., hIL10B may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) Compared to the sequence of NO:4, it has an N-terminal deletion at SPGQGTQSEN (SEQ ID NO:21).

[0162] In certain embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A containing a sequence having the amino acid substitution T100L, and hIL10B containing a sequence having the amino acid substitution T100L, wherein hIL10B further has an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4, where n is 1 and L is GGGSGSGSGSG (SEQ ID NO:19). In a particular embodiment, the fusion hIL10 polypeptide has the sequence of SEQ ID NO:36.

[0163] In certain embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A containing a sequence having the amino acid substitution T100L and hIL10B containing a sequence having the amino acid substitution T100L, where n is 1 and L is T. In a particular embodiment, the fusion hIL10 polypeptide has the sequence SEQ ID NO:37.

[0164] In certain embodiments, the fusion hIL10 polypeptide having the formula (hIL10A)-L n -(hIL10B) can comprise hIL10A comprising a sequence having the amino acid substitution T100L and hIL10B comprising a sequence having the amino acid substitution T100L, and hIL10B also has a N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4, n is 1, and L is NQMFDQKYDDP (SEQ ID NO:20). In certain embodiments, the fusion hIL10 polypeptide has the sequence of SEQ ID NO:38.

[0165] In certain embodiments, the fusion hIL10 polypeptide having the formula (hIL10A)-L n -(hIL10B) can comprise hIL10A comprising a sequence having the amino acid substitution T100L and hIL10B comprising a sequence having the amino acid substitution T100L, and n is 0. In certain embodiments, the fusion hIL10 polypeptide has a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the sequence of SEQ ID NO:44.

[0166] In certain embodiments, the fusion hIL10 polypeptide having the formula (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains an amino acid substitution E96Q and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and hIL10B, which contains an amino acid substitution E96Q and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and one or both of hIL10A and hIL10B may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4 (e.g., hIL10B may have an N-terminal deletion of SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4. NO:21) has an N-terminal deletion.

[0167] In certain embodiments, (hIL10A)-L n A fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A, which contains an amino acid substitution T100L and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) compared to the sequence of SEQ ID NO:4, and hIL10B, which contains an sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) compared to the sequence of SEQ ID NO:4.

[0168] In certain embodiments, (hIL10A)-L nA fusion hIL10 polypeptide having the formula -(hIL10B) may include hIL10A containing a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) compared to the sequence of SEQ ID NO:4, and hIL10B containing a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) compared to the sequence of SEQ ID NO:4, and one or both of hIL10A and hIL10B may have an N-terminal deletion of SPGQGTQ (SEQ ID NO:22) or SPGQGTQSEN (SEQ ID NO:21) compared to the sequence of SEQ ID NO:4 (e.g., hIL10A may have an N-terminal deletion of SPGQGTQ (SEQ ID NO:22) or SPGQGTQSEN (SEQ ID NO:21)). Compared to the sequence of NO:4, it has an N-terminal deletion at SPGQGTQSEN (SEQ ID NO:21).

[0169] Examples of fusion hIL10 polypeptides and the nucleic acid sequences they encode are shown in Table 2 below. Linker sequences (L) are highlighted in bold. In some embodiments, the fusion hIL10 polypeptide comprises one of the polypeptide sequences in Table 2 and optionally has the linker shown, or a different linker as described herein, or lacks a linker. In some embodiments, a His tag can be fused to the N-terminus of each of the fusion hIL10 polypeptide sequences listed below, with or without a linker; that is, GGSHHHHHHHH (SEQ ID NO: 23) can be fused to the N-terminus of the fusion hIL10 polypeptides listed below. The fusion hIL10 polypeptides listed in Table 2 are prepared using mature hIL10 monomers from which the 18-amino acid signal sequence (SEQ ID NO: 5) has been cleaved.

[0170] In some aspects, this disclosure corresponds to SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 119, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183 A polypeptide selected from the group consisting of 184, 185, and 186 has sequence identity of 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%, with the formula (hIL10A)-L n -(hIL10B) provides a fused IL10 polypeptide.

[0171] This disclosure provides fused IL10 polypeptides having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100%) sequence identity to any one of the polypeptide sequences in Table 2. In some embodiments, this disclosure provides fused IL10 polypeptides 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 any one of the polypeptide sequences in Table 2, and optionally having the linkers shown, or having different linkers as described herein, or lacking linkers.

[0172] In some aspects, the formula is (hIL10A)-L nThe fused IL10 polypeptide, which is -(hIL10B), is selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 119.

[0173] (Table 2) Exemplary fused IL10 polypeptides and the nucleic acids encoding them TIFF2026521391000014.tif187148TIFF2026521391000015.tif187148TIFF2026521391000016.tif187148TIFF20265213910 00017.tif187148TIFF2026521391000018.tif187148TIFF2026521391000019.tif187148TIFF2026521391000020.tif187148 TIFF2026521391000021.tif187148TIFF2026521391000022.tif187148TIFF2026521391000023.tif187148TIFF20265213910 00024.tif187148TIFF2026521391000025.tif187148TIFF2026521391000026.tif187148TIFF2026521391000027.tif183148

[0174] This disclosure relates to a polypeptide 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 any one of the polypeptide sequences in Table 2, with the formula (hIL10A)-L n-Provided is a fusion IL10 polypeptide that is (hIL10B). The present disclosure relates to 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 any one of the polypeptide sequences in Table 2A, and the formula is (hIL10A)-L n -Provided is a fusion IL10 polypeptide that is (hIL10B), and in some embodiments, the formula is (hIL10A)-L n -The fusion IL10 polypeptide that is (hIL10B) has at least 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 a polypeptide selected from the group consisting of SEQ ID NO: 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, and 171. In some embodiments, the formula is (hIL10A)-L n -The fusion IL10 polypeptide that is (hIL10B) is a polypeptide selected from the group consisting of SEQ ID NO: 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, and 171. In some embodiments, the formula is (hIL10A)-L n -The fusion IL10 polypeptide that is (hIL10B) is SEQ ID NO: 159. In some embodiments, the formula is (hIL10A)-L n -The fusion IL10 polypeptide that is (hIL10B) is SEQ ID NO: 160. In some embodiments, the formula is (hIL10A)-L n -The fusion IL10 polypeptide that is (hIL10B) is SEQ ID NO: 165. In some embodiments, the formula is (hIL10A)-L nThe fused IL10 polypeptide, which is -(hIL10B), has SEQ ID NO: 170.

[0175] (Table 2A) TIFF2026521391000028.tif84148TIFF2026521391000029.tif194148TIFF2026521391000030.tif194148TIFF2026521391000031.tif121148

[0176] As described herein, the N-terminus of the molecule being a fused hIL10 polypeptide is recombinantly produced in bacterial cells by direct expression (i.e., not as a fusion protein), and the naturally occurring N-terminal serine may be deleted to provide efficient cleavage of the N-terminal methionyl residue. This disclosure relates to a molecule 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 any one of the polypeptide sequences in Table 2B, with the formula (hIL10A)-L n The disclosure provides a fusion IL10 polypeptide having the formula (hIL10A)-L, which has 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 any one of the polypeptide sequences in Table 2B. n Provides a fusion IL10 polypeptide whose formula is -(hIL10B). In some embodiments, the formula is (hIL10A)-L nA fusion IL10 polypeptide having the formula (hIL10B) is 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 selected from the group consisting of SEQ ID NO: 172, 173, 174, 175, 179, 180, 181, 182, 183, 184, 185, and 186. In some embodiments, the formula is (hIL10A)-L n The fused IL10 polypeptide with the formula -(hIL10B) has SEQ ID NO:174. In some embodiments, the formula is (hIL10A)-L n The fused IL10 polypeptide with the formula -(hIL10B) has SEQ ID NO: 175. In some embodiments, the formula is (hIL10A)-L n The fused IL10 polypeptide with formula -(hIL10B) has SEQ ID NO: 180. In some embodiments, the formula is (hIL10A)-L n The fused IL10 polypeptide with the formula -(hIL10B) has SEQ ID NO:185. In some embodiments, the formula is (hIL10A)-L n The fused IL10 polypeptide, which is -(hIL10B), is selected from the group consisting of SEQ ID NO: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, and 186.

[0177] (Table 2B) TIFF2026521391000032.tif121148TIFF2026521391000033.tif194148TIFF2026521391000034.tif194148TIFF2026521391000035.tif84148

[0178] Examples of fused mouse IL10 polypeptides are shown in the table below, where linkers are shown in bold. In some embodiments, a His tag can be fused to the N-terminus of each of the fused mRNA polypeptide sequences listed below, with or without a linker; that is, GGSHHHHHHHH (SEQ ID NO: 23) can be fused to the N-terminus of the fused mRNA polypeptides listed below.

[0179] (Table 3) Exemplary mouse fused IL10 polypeptide and the nucleic acid encoding it TIFF2026521391000036.tif195145TIFF2026521391000037.tif191145TIFF20265213910 00038.tif186145TIFF2026521391000039.tif195145TIFF2026521391000040.tif186145

[0180] Furthermore, the hIL10 mutain and fusion hIL10 polypeptides containing hIL10 mutain of this disclosure may exhibit differential expression or thermal stability depending on specific mutations incorporated into the hIL10A subunit or the hIL10B subunit. The hIL10 mutain and fusion hIL10 polypeptides containing hIL10 mutain (including the T100L, N21K, M22A, or R24E mutations) may show increased expression or thermal stability compared to hIL10 mutain having substitution mutations at other amino acid positions or different amino acid substitutions at the same position.

[0181] 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.

[0182] 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.

[0183] In some embodiments, the fusion hIL10 polypeptide of this disclosure exhibits cell type biased activity compared to wild-type IL10. As used herein, the term “biased” as used in relation to the fusion IL10 polypeptide is used to indicate that the fusion IL10 polypeptide exhibits a percentage increase in the level of wild-type IL10 activity in a first cell type compared to the level of wild-type IL10 activity in a second cell type, compared to the level of wild-type IL10 activity in a second cell type. 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 one embodiment, the first cell type is LPS-activated human myeloid cells. In some embodiments, the second cell type is a T cell.

[0184] In some embodiments, the fusion IL10 polypeptide of the Disclosure inhibits 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 fusion hIL10 polypeptide of the Disclosure retains the immunosuppressive function of wild-type hIL10, e.g., inhibits the production of inflammatory cytokines, whereas the immunostimulatory function of wild-type hIL10, e.g., CD8 + It reduces IFNγ production by T cells. For example, in some embodiments, the fused hIL10 polypeptide of this disclosure retains activity equivalent to wild-type hIL10 to suppress myeloid cell activation (e.g., as assessed by increased STAT3-mediated signaling in myeloid cells), but its activation is substantially reduced in PBMCs, T cells, B cells, and NK cells (e.g., as assessed by reduced IFNγ production).

[0185] In some embodiments, the fused IL10 polypeptide of the present disclosure is an hIL10 partial agonist.

[0186] Relative STAT3 induction As mentioned above, the interaction between IL10 and the IL10 receptor results in intracellular signaling characterized by enhanced intracellular production of phosphorylated STAT3 (phosphor-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.

[0187] In one embodiment, the fusion hIL10 polypeptide is a biased hIL10 partial agonist, the first cell type is activated human myeloid cells, and 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 fusion hIL10 polypeptide 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 some embodiments, the relative activation of STAT3 signaling by the fusion hIL10 polypeptide described herein in the first cell type versus the second cell type is different from the relative activation of STAT3 signaling by wild-type human IL10 or wild-type mouse IL10 in the first cell type versus the second cell type. In some embodiments, the level of intracellular phospho-STAT3 induced in human myeloid cells in response to contact between myeloid cells and an effective amount of fused hIL10 polypeptide 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 fused hIL10 polypeptide as human lymphocyte cells. In one embodiment, the ratio of the level of STAT3 signaling induced in myeloid cells in response to contact between myeloid cells and fused hIL10 polypeptide to the level of STAT3 signaling induced in lymphocyte cells in response to contact between lymphocytes and fused hIL10 polypeptide is different from (or greater than) the ratio of the level of STAT3 signaling induced in myeloid cells in response to contact between myeloid cells and wild-type hIL10 to the level of STAT3 signaling induced in lymphocytes in response to contact between lymphocytes and wild-type hIL10. In some embodiments, the ratio of the activity of fused hIL10 polypeptide in human bone marrow cells (as determined by intracellular phospho-STAT3 levels) to the activity of fused hIL10 polypeptide in human lymphocytes is greater than the ratio of the activity of wild-type human IL10 in human bone marrow cells to the activity of wild-type human IL10 in human lymphocytes. In some embodiments, the bone marrow 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.

[0188] In some embodiments, the fused hIL10 polypeptide of this disclosure is equivalent to the pSTAT3 E of wild-type hIL10 in bone marrow cells. max pENTER3 E max (See, for example, Figure 2A). In some embodiments, the fused hIL10 polypeptide of the Disclosure exhibits reduced STAT3-mediated signaling in lymphocytes such as T cells, B cells, or NK cells compared to wild-type hIL10. In some embodiments, the fused hIL10 polypeptide of the Disclosure exhibits 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 has within the lymphocyte. In some embodiments, the fused hIL10 polypeptide has pSTAT3 E of the wild-type IL10 polypeptide or parent IL10 polypeptide within the lymphocyte. max Less than 70% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, or less than 30%) and more than 20% of pSTAT3 E max This is brought about within the lymphocyte. In some embodiments, the lymphocyte is selected from CD8+ T cells, CD4+ T cells, B cells, or NK cells.

[0189] Relative pro-inflammatory activity and anti-inflammatory activity In some embodiments, the fused IL10 polypeptide exhibits (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, “a significant level of at least one anti-inflammatory property” means that the Emax of the fused IL10 polypeptide with respect to such an anti-inflammatory property is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the Emax level of such an anti-inflammatory property exhibited by wild-type IL10, when determined in the test system. Examples of anti-inflammatory properties that may be measured in the test system include, but are not limited to, (a) suppression of ILβ expression and / or secretion by activated human myeloid cells, (b) suppression of IL6 expression and / or secretion by activated human myeloid cells, and (c) suppression of TNFα expression and / or secretion by activated human myeloid cells. In some embodiments, activated human bone marrow cells are obtained by isolating human monocytes from the buffy coat of a centrifuged anticoagulant-treated human blood sample according to procedures well known in the art, and activating the isolated monocytes by contacting them with lipopolysaccharide [LPS]. The levels of ILβ, IL6, and TNFα expressed and / or secreted by the activated monocytes can be determined by immunoassay or flow cytometry according to procedures well known in the art. Protocols for evaluating the suppression of ILβ, IL6, and TNFα expression and / or secretion by LPS-activated human monocytes are provided in the examples herein.

[0190] In some embodiments, “a markedly reduced level of at least one pro-inflammatory property” means that the Emax of the fusion IL10 polypeptide 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. Protocols 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 are provided in the examples.

[0191] In some embodiments, the fused hIL10 exhibits 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 hIL10, where (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 greater than 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, (ii) suppression of hIL6 expression and / or secretion in LPS-activated human monocytes, or (iii) h (b) The group is selected from the group consisting of suppression of TNFα expression and / or secretion, and (b) the significantly 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.

[0192] In some embodiments, the fusion hIL10 polypeptide of the Disclosure, comprising a polypeptide of formula (1) or formula (2), is a biased hIL10 partial agonist exhibiting 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 greater than 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, or (ii) expression and / or hIL6 expression in LPS-activated human monocytes. (iii) Suppression of secretion, or (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.

[0193] In some embodiments, the fused hIL10 polypeptide is a partial agonist, and (a) the Emax of the fused IL10 polypeptide is greater than 30% of the Emax of wild-type hIL10 in an anti-inflammatory activity assay selected from the group consisting of (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, and (b) the Emax of the fused IL10 polypeptide is less than 10% of the Emax of wild-type hIL10 in an anti-inflammatory activity assay selected from the group consisting of (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (ii) 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.

[0194] In some embodiments, the fused hIL10 polypeptide is a partial agonist, and (a) the Emax of the fused hIL10 polypeptide is greater than 50% of the Emax of wild-type hIL10 in an anti-inflammatory activity assay selected from the group consisting of (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, and (b) the Emax of the fused hIL10 polypeptide is less than 20% of the Emax of wild-type hIL10 in an anti-inflammatory activity assay selected from the group consisting of (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (ii) 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.

[0195] In some embodiments, the fused hIL10 polypeptide is a partial agonist, and (a) the Emax of the fused hIL10 polypeptide is greater than 50% of the Emax of wild-type hIL10 in an anti-inflammatory activity assay selected from the group consisting of (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, and (b) the Emax of the fused hIL10 polypeptide is less than 10% of the Emax of wild-type hIL10 in an anti-inflammatory activity assay selected from the group consisting of (i) suppression of IFNγ expression and / or secretion by activated human CD8+ T cells, (ii) 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.

[0196] Modifications to provide additional functionality In some embodiments, hIL10 mutein may comprise a functional domain of a chimeric polypeptide. The human IL10 mutein fusion protein 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 hIL10 mutein and a nucleic acid sequence encoding hIL10 mutein in frame, wherein the sequence optionally further comprises a nucleic acid sequence encoding a linker polypeptide or spacer polypeptide in frame.

[0197] In other embodiments, hIL10 mutein 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.

[0198] In some embodiments, hIL10 mutain, which optionally incorporates a 1-40 (or 2-20, or 5-20, or 10-20) amino acid linker molecule between the hIL10 mutain sequence and the sequence of the targeting domain of the fusion protein, is 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.

[0199] In other embodiments, a chimeric polypeptide comprising hIL10 mutaine and an antibody or its antigen-binding moiety can be generated. For example, a chimeric polypeptide may be used to localize a chimeric protein to a specific subset of cells or target molecules. The antibody or antigen-binding component of the chimeric protein may serve as the targeting moiety. 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, equine antibodies, camelid antibodies, and human antibodies. The term antibody includes "heavy chain antibodies" and single-domain antibodies (sdAb), e.g., "VHH," typically obtained from immunization of camelids (camels, llamas, and alpacas), as described in more detail below in the definition of "VHH," for example, 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, the 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, and Lyve1. Methods for generating cytokine-antibody chimeric polypeptides are described, for example, in U.S. Patent No. 6,617,135.

[0200] Association with carrier molecules to increase the duration of action The fusion hIL10 polypeptides described herein may be modified to provide in vivo lifetime extension and / or duration of action extension in a subject. In some embodiments, the fusion hIL10 polypeptide is conjugated to one or more carrier molecules to provide desired pharmacological properties, such as extension of half-life. In some embodiments, the fusion hIL10 polypeptide is covalently bound to the Fc domain of IgG, albumin, a water-soluble polymer or other molecule to extend its half-life, such as glycosylation, acylation, etc., as known in the art. In some embodiments, fused hIL10 polypeptides modified to provide an extended duration of action in mammalian subjects have a half-life in mammals 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.

[0201] Modifications of fusion hIL10 polypeptides to provide an extended duration of action in mammalian subjects include (but are not limited to) the following: • Conjugation of fused hIL10 polypeptides to one or more protein carrier molecules. Optionally, conjugation of the fused hIL10 polypeptide to a protein carrier molecule in the form of a fusion protein having an additional polypeptide sequence (e.g., a fused hIL10 polypeptide-Fc fusion), and • Conjugation to polymers (e.g., water-soluble polymers for providing PEGylated IL10 polypeptides).

[0202] 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 fusion hIL10 polypeptide. For example, the fusion hIL10 polypeptide 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.

[0203] Examples of protein carrier molecules that can be covalently bound to a fused hIL10 polypeptide to provide an extended 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.

[0204] Fc fusion In some embodiments, the fusion IL10 polypeptide can be conjugated to an Fc polypeptide. In some embodiments, the fusion IL10 polypeptide is conjugated to one Fc polypeptide of an Fc dimer. In some embodiments, the fusion IL10 polypeptide is conjugated to both Fc polypeptides of an Fc dimer. Schematic diagrams of four exemplary configurations of the Fc-conjugated fusion IL10 polypeptide are shown in Figures 18A and 18B of the attached drawings. As illustrated in Figures 18A and 18B, the IL10 fusion polypeptide can be covalently attached to the N-terminus of one Fc polypeptide of an Fc dimer that has been modified to facilitate heterodimerization using a knob-into-hole (KiH) modification to the Fc polypeptide, which can then be conjugated to either an Fc polypeptide containing a “hole” amino acid substitution (Figure 18A) or a “knob” amino acid substitution (Figure 18B). In addition, as illustrated in Figure 18B, two different IL10 fusion polypeptides can be conjugated to Fc polypeptides of Fc dimers, respectively, in which the Fc domain is modified to promote heterodimerization. Alternatively, as illustrated in Figure 18D, two identical IL10 fusion polypeptides can be conjugated to Fc polypeptides of Fc dimers, respectively, in which they are not modified to promote heterodimerization.

[0205] The Fc polypeptide may be an Fc domain derived from hIgG1, hIgG2, hIgG3, or hIgG4, or a variant thereof. In some embodiments, the Fc polypeptide includes a sequence modified from the wild-type Fc polypeptide sequence to reduce effector function. In some embodiments, the Fc dimer Fc polypeptide may be modified to promote heterodimerization.

[0206] The "Fc region" useful for preparing Fc fusions may be a natural or synthetic polypeptide homologous to the IgG C-terminal domain produced by papain digestion of IgG. 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 in which it is a part. Furthermore, the full-length Fc region or a fragmented Fc region may be a variant of the wild-type molecule.

[0207] As shown, linking the fusion hIL10 polypeptide to the Fc subunit may incorporate a linker molecule between the fusion hIL10 polypeptide and the Fc subunit. In some embodiments, the fusion hIL10 polypeptide 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 fusion hIL10 polypeptide is linked to the Fc domain using the human IgG1 hinge domain.

[0208] 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 polymer, alanine-serine polymer, glycine-serine polymer (e.g., (GmSo)n (SEQ ID NO: 129), (GSGGS)n (SEQ ID NO: 130), (GmSoGm)n (SEQ ID NO: 131), (GmSoGmSoGm)n (SEQ ID NO: 132), (GSGGSm)n (SEQ ID NO: 133), (GSGSmG)n (SEQ ID NO: 134), and (GGGSm)n (SEQ ID Examples include NO:135), as well as combinations thereof, where m, n, and o are integers of 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, and other flexible linkers.

[0209] In some embodiments, the amino acid sequence of an Fc polypeptide conjugated to a fusion hIL10 polypeptide can be modified to reduce effector function. In some embodiments, the Fc polypeptide can be modified to substantially reduce its binding to Fc receptors (FcyR and FcR), thereby reducing or eliminating antibody-directed cytotoxicity (ADCC) effector function. Modifications of Fc polypeptides 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 polypeptide 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 polypeptide may contain the amino acid substitution E233P / L234V / L235A / ΔG237 (known in scientific literature as the PVAdelG mutation).

[0210] In some embodiments, the Fc polypeptide 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).

[0211] In some embodiments, the amino acid sequence of the Fc polypeptide may be further 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 polypeptide 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 and result in disulfide bonds between Fc domains.

[0212] In some embodiments, the amino acid sequence of the Fc polypeptide conjugated to the fusion hIL10 polypeptide may be further modified to eliminate an N-linked or O-linked glycosylation site. In particular, deglycosylated variants of IgG1 subclass Fc polypeptides are known to have poor mediators of effector function (Jefferies et al. 1998, Immol. Rev., vol. 163, 50-76). Glycosylation at position 297 (EU numbering) has been shown to contribute to effector function (Edelman, et al. (1969) PNAS (USA) 63:78-85). In some embodiments, the Fc polypeptide includes one or more modifications to eliminate an N-linked or O-linked glycosylation site. Examples of modifications at N297 to eliminate a glycosylation site in the Fc polypeptide include amino acid substitutions N297Q and N297G. In some embodiments, the Fc polypeptide is hIgG4 Fc containing amino acid substitutions selected from the group consisting of N297Q and N297G.

[0213] In some embodiments, the fused hIL10Fc polypeptide 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 the cysteine ​​residue. In some embodiments, PEGylation of the fused hIL10 polypeptide is provided to a naturally occurring cysteine ​​residue at position 220 (C220, EU numbering) of the upper hinge region of the Fc polypeptide.

[0214] In some embodiments, a fusion hIL10 polypeptide can be conjugated to a single Fc polypeptide of an Fc dimer, where the first and second Fc polypeptides of the Fc dimer are modified to promote heterodimerization (Figures 18A and 18B). In some embodiments, a fusion hIL10 polypeptide can be conjugated to both Fc polypeptides of an Fc dimer, where the first and second Fc polypeptides of the Fc dimer are modified to promote heterodimerization, and each of the fusion hIL10 polypeptides may be the same or different (Figure 18C). Various techniques for promoting heterodimerization of the Fc domain have been established. See, for example, Kim, et al., U.S. Patent No. 11,087,249, published 3 August 2021. In some embodiments, modifications for promoter heterodimerization of the first and second Fc polypeptides are the HF-TA and HA-TF mutations described in Moore, et al (2011) mAbs 3(6):546-557. The HF-TA method uses an S364H / T394F substitution on one Fc monomer and a Y349T / F405A substitution on the complementary Fc polypeptide. The (HA-TF) method uses an S364H / F405A substitution on one Fc polypeptide and a Y349T / T394F substitution on the complementary Fc polypeptide. Alternatively, the first and second Fc polypeptides are modified to promote heterodimerization by the ZW1 heterodimerization method using a T350V / L351Y / F405A / Y407V substitution on one Fc polypeptide and a T350V / T366L / K392L / T394W substitution on the complementary Fc polypeptide. Von Kreudenstein, et al (2013) mAbs, 5(5):646-654. Alternatively, the first and second Fc polypeptides are modified to promote heterodimerization by the EW-RVT heterodimerization method using a K360E / K409W substitution on one Fc polypeptide and a Q347R / D399V / F405T substitution on the complementary Fc polypeptide. Choi, et al (2015) Molecular Immunology 65(2):377-83.

[0215] In one embodiment, the first and second Fc polypeptides are modified to facilitate heterodimerization by using a “knob-into-hole (abbreviated as KiH)” modification as exemplified herein. The KiH modification comprises one or more amino acid substitutions in the first Fc polypeptide that create a bulky “knob” domain on the first Fc polypeptide, and one or more amino acid substitutions on the second Fc polypeptide that create a complementary pocket or “hole” that accepts the “knob” of the first Fc monomer. Various amino acid substitutions have been established to create complementary knob and hole Fc monomers. For example, Ridgway, et al (1996) Protein Engineering 9(7):617-921; Atwell, et al (1997) J.Mol.Biol.270:26-35; Carter, et al., U.S. Patent No. 5,807,706, issued September 15, 1998; Carter, et al., U.S. Patent No. 7,695,936, issued April 13, 2010; Zhao et al., "A new approach to produce IgG4-like bispecific antibodies," Scientific Reports 11:18630(2021); Cao et al., "Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody," and Liu et al., "Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds," Frontiers in Immunology. See 8:38,2017.In some embodiments, the Fc dimer comprises two Fc polypeptides in which the CH3 domain of a first Fc polypeptide is modified by a bulky residue at (EU numbering) position 366 (e.g., T366W), creating a “knob” and substitution; and a second Fc polypeptide containing one or more substitutions at complementary residues in the CH3 domain of a second Fc polypeptide, such as amino acid substitutions including T366S, L368A and / or Y407V, creating a pocket or “hole” that accepts the bulky residue. In one embodiment, the Fc1 monomer of formula 1 is a “knob” modified Fc monomer containing the amino acid substitution T366W, and the Fc2 monomer of formula 2 is a “hole” modified Fc containing the set of amino acid substitutions T366S / L368A / Y407V. In one embodiment, the first Fc polypeptide of the Fc dimer is a “whole” modified Fc monomer containing the amino acid substitution set T366S / L368A / Y407V, and the second Fc polypeptide of the Fc dimer is a “knob” modified Fc monomer containing the amino acid substitution T366W.

[0216] In some embodiments, the fusion hIL10 polypeptide Fc conjugates of the present disclosure are provided as complementary heterodimer pairs of fusion hIL10 Fc polypeptides, wherein the first and second fusion hIL10 polypeptide Fc polypeptides are linked by at least one disulfide bond. In some embodiments, the incorporation of a disulfide bond between the first and second fusion hIL10 polypeptide Fc polypeptides can be achieved by cysteine ​​substitution at specific points within the first and second Fc polypeptides. In one embodiment, to provide a disulfide bond between S354C of the first Fc polypeptide and Y349C of the second Fc polypeptide, the Fc polypeptide of the first fusion hIL10 Fc polypeptide is derived from the Fc domain of hIgG1 containing the amino acid substitution S354C (EU numbering), and the second fusion hIL10 Fc polypeptide domain is derived from the Fc domain of hIgG1 containing the amino acid substitution Y349C (EU numbering). Alternatively, in order to provide a disulfide bond between S354C of the first Fc polypeptide and Y349C of the second Fc polypeptide, the first Fc polypeptide is derived from the Fc domain of hIgG1 containing the amino acid substitution Y349C (EU numbering), and the second Fc polypeptide is derived from the Fc domain of hIgG1 containing the amino acid substitution S354C (EU numbering).

[0217] In some aspects, this disclosure relates to formula #1: IL10FP-L1 a -UH1-Fc1 [1] The first polypeptide and formula #2: UH2-Fc2 [2] We provide a heterodimerized IL10 polypeptide Fc comprising a second polypeptide, During the ceremony, • hIL10FP is a fused hIL10 polypeptide (e.g., formula (hIL10A)-L) of the present disclosure. n -(hIL10B) is a fused hIL10 polypeptide, L1 is the linker, and a is selected independently of 0 (non-existent) or 1 (existent). UH1 and UH2 are upper hinge domains of human immunoglobulins, independently selected from the group consisting of the upper hinge domains of IgG1, IgG2, IgG3, and IgG4, and optionally contain the amino acid substitution C220S (EU numbering). Fc1 is a polypeptide comprising a lower hinge, CH2 domain, and CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and contains one or more amino acid substitutions that promote heterodimerization with Fc2, and Fc2 is a polypeptide comprising a lower hinge, CH2 domain, and CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and contains one or more amino acid substitutions that promote heterodimerization with Fc1. Optionally, the polypeptide of formula [1] and the polypeptide of formula [2] are linked by at least one interchain disulfide bond.

[0218] The heterodimerized IL10 polypeptide Fc of this disclosure is a heterodimer comprising polypeptides of formulas [1] and [2], each incorporating the upper hinge region of a human immunoglobulin molecule, respectively. The term “upper hinge” or “UH” refers to the amino acid sequence corresponding to amino acid residues 216–220 (EU numbering) of a human immunoglobulin molecule. In some embodiments, the upper hinge region is a naturally occurring upper hinge region of human immunoglobulin, selected from the upper hinge domains of human IgG1, human IgG2, human IgG3, and human IgG4. In some embodiments, the upper hinge region is the upper hinge region of human IgG1 immunoglobulin. In some embodiments, the upper hinge region is the upper hinge region of human IgG1 immunoglobulin comprising the pentameric amino acid sequence: EPKSC (SEQ ID NO: 121).

[0219] In some embodiments, the upper hinge region contains an unpaired cysteine ​​residue at position 220 (EU numbering), which typically binds to cysteine ​​on the light chain in the complete immunoglobulin molecule. When only the Fc domain containing the hinge domain is used, the unpaired cysteine ​​within the hinge domain leads to the possibility of improper disulfide bond formation. Consequently, in some embodiments, the cysteine ​​at position 220 (C220, numbered according to EU numbering) is replaced with an amino acid that does not promote disulfide bond formation. In some embodiments, the Fc domain contains a C220S mutation with the amino acid sequence EPKSS (SEQ ID NO: 122).

[0220] Fc1 and Fc2 The heterodimerized IL10 polypeptide Fc of this disclosure is a heterodimer comprising polypeptides of formulas [1] and [2], each incorporating Fc regions (Fc1 and Fc2) of a human immunoglobulin molecule modified to promote heterodimerization.

[0221] As used herein, the terms “Fc,” “Fc monomer,” and “Fc polypeptide” are used interchangeably to refer to the monomeric polypeptide subunit of the Fc dimer. Fc comprises an amino acid sequence (from the amino terminus to the carboxyl terminus) including the lower hinge domain of the human immunoglobulin molecule, as well as the CH2 and CH3 domains. In some embodiments, the Fc monomer is a polypeptide comprising the lower hinge domain of the human immunoglobulin molecule domain, as well as the CH2 and CH3 domains, of the hinge domains of human IgG1, human IgG2, human IgG3, and human IgG4. The CH2 domain of hIgG1 corresponds to amino acid residues 231-340 (EU numbering) and is provided as SEQ ID NO: 128. The CH3 domain of hIgG1 corresponds to amino acid residues 341-447 (EU numbering) and is provided as SEQ ID NO: 128.

[0222] The polypeptides of formulas [1] and [2] each incorporate the lower hinge region of a human immunoglobulin, respectively. As used herein, the terms “lower hinge” or “LH” refer to the amino acid sequence corresponding to amino acid residues 221–229 (EU numbering) of a human immunoglobulin molecule. In some embodiments, the lower hinge region is a naturally occurring lower hinge region of a human immunoglobulin, selected from the LH regions of the lower hinge domains of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the lower hinge region is the lower hinge region of human IgG1 immunoglobulin. In some embodiments, the lower hinge region is the lower hinge region of human IgG1 immunoglobulin containing the decameric amino acid sequence: DKTHTCPPCP (SEQ ID NO: 127).

[0223] In some embodiments, Fc1 and Fc2 are derived from polypeptides corresponding to amino acids 221-447 (EU numbering) of human IgG1 immunoglobulin, as shown below. TIFF2026521391000041.tif55128

[0224] As shown in the sequence above, the C-terminal residue of the wild-type form of the IgG1 Fc domain is a lysine residue called K447 according to EU numbering. K447 is inconsistently removed by producer cells during recombinant production. As a result, the recombinant Fc monomer population can be heterogeneous, with some recombinant Fc monomers containing K447 and others not. Thus, such inconsistent proteolytic processing by producer cells can result in a heterogeneous population of hIL10 Fc. Typically, such heterogeneity of the active pharmaceutical component should be avoided, especially in relation to human pharmaceuticals. As a result, in addition to the fact that modifications to the Fc monomer sequence promote heterodimerization, the Disclosure provides an Fc monomer comprising a deletion of C-terminal K447, or further deletions of G446 and K447, and a nucleic acid sequence encoding an Fc monomer comprising (a) a deletion of the lysine residue at position 447 (abbreviated as K447, EU numbering, ΔK447, or des-K447), or (b) a deletion of both glycine at position 456 (G446, EU numbering, abbreviated as des-G446) and K447 (this double deletion of G446 and K447 is referred to herein as des-G446 / des-K447, or ΔG446 / ΔK447).

[0225] As provided by formulas [1] and [2] above, the Fc1 monomer and Fc2 monomer of dimer Fc contain amino acid substitutions that promote heterodimerization between Fc1 and Fc2.

[0226] In some embodiments, the fusion hIL10 polypeptide Fc conjugate of the present disclosure is covalently bonded via one or more, optionally two or more, optionally three or more disulfide bonds, or optionally four or more disulfide bonds between the side chains of the following groups of the cystine pair: (a) C96 of hP35 and C199 of hP40M; (b) between C226 of the first Fc monomer and C226 of the second Fc monomer; (c) between C229 of the first Fc monomer and C229 of the second Fc monomer; and (d) between S354C of the first Fc domain containing the S354C amino acid substitution and Y349C of the second Fc domain containing the Y349C amino acid substitution.

[0227] Albumin carrier molecule In some embodiments, the fusion hIL10 polypeptide is conjugated to an albumin molecule known in the art (e.g., human serum albumin) to facilitate the extension of in vivo exposure. In some embodiments, the fusion hIL10 polypeptide is conjugated to albumin via a chemical bond or expressed as a fusion protein with an albumin molecule (referred to herein as the “fusion hIL10 polypeptide-albumin fusion”). The term “albumin” as used in relation to the fusion hIL10 polypeptide-albumin fusion includes albumins such as human serum albumin (HSA), cynomolgus monkey serum albumin, and bovine serum albumin (BSA). In some embodiments, the 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 fusion hIL10 polypeptide internally (see, for example, U.S. Patent Nos. 5,876,969 and 7,056,701). In the HAS-fusion hIL10 polypeptide intended 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 a fusion hIL10 polypeptide 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 fusion hIL10 polypeptide and the albumin molecule, the fusion hIL10 polypeptide-albumin complex may be provided as a fusion protein comprising an albumin polypeptide sequence and a fusion hIL10 polypeptide recombinantly expressed in a host cell as a single polypeptide chain, and optionally including a linker molecule between albumin and the fusion hIL10 polypeptide. Such a fusion protein 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 sequences encoding the fusion protein are 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 sol-formed from the host cell culture by techniques well known in the art.

[0228] Polymer carrier In some embodiments, the extension of the in vivo duration of action of the fused hIL10 polypeptide can be achieved by conjugation to one or more polymer support molecules, such as an XTEN polymer or a water-soluble polymer.

[0229] XTEN Conjugate The fused hIL10 polypeptide may further contain XTEN polymers. XTEN polymers conjugated to the fused hIL10 polypeptide (chemically or as fusion proteins) provide a duration extension similar to PEGylation and can be produced as recombinant fusion proteins in E. coli. XTEN polymers suitable for use in combination with the fused hIL10 polypeptide 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 hIL2 mutein.

[0230] Water-soluble polymers In some embodiments, the fused hIL10 polypeptide can be conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful for carrying out the disclosure include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymer of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyolefin alcohols, poly-alpha-hydroxy acids), polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0231] PEGylation In some embodiments, the fused hIL10 polypeptide can be conjugated to one or more polyethylene glycol molecules, or "PEGylated." The method or site of PEGylation to the binding molecule can vary, but in certain embodiments, PEGylation does not alter, or alters minimally alters, the activity of the binding molecule.

[0232] 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.

[0233] In some embodiments, selective PEGylation of the fused hIL10 polypeptide may be used, for example, by incorporating non-natural amino acids having side chains to promote selective PEG conjugation. The specific PEGylation site may be selected so that PEGylation of the binding molecule does not affect its binding to the target receptor.

[0234] 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.

[0235] 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.

[0236] In some embodiments, the present disclosure is based on the following formula: PEG-L2 m -[(hIL10A)-L n -(hIL10B)] We provide a fused human IL10 (hIL10) polypeptide. During the ceremony, PEG is a linear or branched polyethylene glycol molecule having a molecular weight of approximately 10 kD to approximately 80 kD. L2 is a polypeptide or chemical linker, where m=0 (non-existent) or 1 (existent). hIL10A contains amino acid substitutions at positions selected from the group consisting of T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96 and R104, numbered according to SEQ ID NO:4 (for example, selected from the group consisting of T100, H14, N18, N21, M22, D25, R32, S93 and E96). Human IL10 mutein optionally containing an N-terminal deletion of one or more amino acids selected from the group consisting of TIFF2026521391000042.tif37150, and optionally containing an additional N-terminal methionine residue. hIL10B contains amino acid substitutions at positions selected from the group consisting of T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96 and R104, numbered according to SEQ ID NO:4 (for example, selected from the group consisting of T100, H14, N18, N21, M22, D25, R32, S93 and E96). Human IL10 mutein optionally containing an N-terminal deletion of one or more amino acids selected from the group consisting of TIFF2026521391000043.tif37150, L is a polypeptide linker consisting of 1 to 30 amino acids, where n=0 (non-existent) or 1 (existent).

[0237] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, a) PEG is a 40kD branched PEG molecule containing two 20kD arms covalently bonded to the N-terminus of hIL10A via an optional aldehyde linker. b) hIL10A is an hIL10 mutaine that contains one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96 and R104 of SEQ ID NO:4 (for example, corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93 and E96), optionally contains an N-terminal deletion corresponding to residues 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 of SEQ ID NO:4, and optionally contains an additional N-terminal methionine residue. c) hIL10B is an hIL10 mutain that contains one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96 and R104 of SEQ ID NO:4 (for example, corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93 and E96), and optionally contains an N-terminal deletion corresponding to residues 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 of SEQ ID NO:4. d) L is a polypeptide linker, and n=0 (does not exist) or 1 (exists).

[0238] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, a) PEG is a 40kD branched PEG molecule containing two 20kD arms covalently bonded to the N-terminus of hIL10A via an optional aldehyde linker. b) hIL10A is an hIL10 mutein selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 and 205, optionally containing an N-terminal deletion corresponding to residues 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 of SEQ ID NO: 4, and optionally containing an additional N-terminal methionine residue. c) hIL10B is an hIL10 mutain selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 and 205, and optionally containing an N-terminal deletion corresponding to residues 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 of SEQ ID NO: 4. d) L is a polypeptide linker, and n=0 (does not exist) or 1 (exists).

[0239] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is a 40kD branched PEG molecule containing two 20kD arms covalently bonded to the N-terminus of hIL10A via an aldehyde linker, (b) hIL10A is an hIL10 mutaine selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, and optionally containing an additional N-terminal methionine residue. (c)hIL10B is an hIL10 mutaine selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 and 205. (d) L is a polypeptide linker, and n=0 (does not exist) or 1 (exists).

[0240] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is a 40 kD branched PEG molecule that optionally contains two 20 kD arms covalently bonded to the N-terminus of hIL10A via an aldehyde linker, and (b) Formula [hIL10A-L n The polypeptide -hIL10B] is a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 119, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, and 186.

[0241] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is a 40 kD branched PEG molecule that optionally contains two 20 kD arms covalently bonded to the N-terminus of hIL10A via an aldehyde linker, and (b) Formula [hIL10A-L n The polypeptide [-hIL10B] is selected from the group consisting of SEQ ID NO: 36, 37, 38, 42, and 44.

[0242] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is optionally covalently bonded to the N-terminus of hIL10A via a linker, with the following formula: TIFF2026521391000044.tif17128 is a 40kD branched PEG molecule containing two 20kD arms, and (b) Formula [hIL10A-L n The polypeptide -hIL10B] is a polypeptide selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 119, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, and 186.

[0243] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-Ln -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, PEG is optionally covalently bonded to the N-terminus of hIL10A via a linker, as follows: TIFF2026521391000045.tif17128 is a 40kD branched PEG molecule containing two 20kD arms. hIL10A is a polypeptide selected from Table 1B. hIL10B is a polypeptide selected from Table 1A. L is a polypeptide linker, where n = 0 (does not exist) or 1 (exists).

[0244] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is optionally covalently bonded to the N-terminus of hIL10A via a linker, with the following formula: TIFF2026521391000046.tif17128 is a 40kD branched PEG molecule containing two 20kD arms, and (b) Formula [hIL10A-L n The polypeptide of -hIL10B] has the following sequence: This is the polypeptide of TIFF2026521391000047.tif31133.

[0245] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is optionally covalently bonded to the N-terminus of hIL10A via a linker, with the following formula: TIFF2026521391000048.tif17128 is a 40kd branched PEG molecule containing two 20kD arms, and (b) Formula [hIL10A-L n The polypeptide of -hIL10B] has the following sequence: This is the polypeptide of TIFF2026521391000049.tif31134.

[0246] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is optionally covalently bonded to the N-terminus of hIL10A via a linker, with the following formula: TIFF2026521391000050.tif17128 is a 40kd branched PEG molecule containing two 20kD arms, and (b) Formula [hIL10A-L n The polypeptide of -hIL10B] has the following sequence: This is the polypeptide of TIFF2026521391000051.tif31134.

[0247] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is optionally covalently bonded to the N-terminus of hIL10A via a linker, with the following formula: TIFF2026521391000052.tif17128 is a 40kD branched PEG molecule containing two 20kD arms, and (b) Formula [hIL10A-L n The polypeptide of -hIL10B] has the following sequence: This is the polypeptide of TIFF2026521391000053.tif31134.

[0248] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-L n -hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is optionally covalently bonded to the N-terminus of hIL10A via a linker, with the following formula: TIFF2026521391000054.tif17128 is a 40kD branched PEG molecule containing two 20kD arms, and (b) Formula [hIL10A-L n The polypeptide of -hIL10B] has the following sequence: This is the polypeptide of TIFF2026521391000055.tif31133.

[0249] In some embodiments, the disclosure provides a “monoPEGylated” fused hIL10 polypeptide.

[0250] In one aspect, the present disclosure has the following structure: PEG-hIL10A-Ln-hIL10B Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is a 40 kD branched PEG molecule optionally linked to the N-terminus of hIL10A via a polypeptide linker or chemical linker. (b) hIL10A and hIL10B are hIL10 mutaines independently selected from hIL10 mutaines that contain one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96 and R104 of SEQ ID NO:4 (for example, corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93 and E96), and (c) L is a polypeptide linker, and n = 0 (does not exist) or 1 (exists).

[0251] In one aspect, the present disclosure has the following structure: PEG-hIL10A-Ln-hIL10B Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is a 40 kD branched PEG molecule optionally linked to the N-terminus of hIL10A via a polypeptide linker or chemical linker. (b) hIL10A and hIL10B are, hIL10A and hIL10B are, SEQ ID Independently selected from hIL10 mutains containing one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96 and R104 of NO:4 (for example, corresponding to residues T100, H14, N18, N21, M22, D25, R32, S93 and E96), wherein one or more amino acid substitutions are selected from the group consisting of N21D, N21E, N21K, M22A, M22S, M22T, M22D, M22W, R24E, D25K, E96K, E96Q, T100E, T100L and T100C, and (c) L is a polypeptide linker, and n = 0 (does not exist) or 1 (exists).

[0252] In one aspect, the present disclosure has the following structure: PEG-hIL10 T100L-hIL10 T100L This provides a PEGylated fused hIL10 polypeptide.

[0253] In one aspect, the present disclosure has the following structure: PEG-[hIL10A-hIL10B] Provides a PEGylated fused hIL10 polypeptide, During the ceremony, (a) PEG is a 40 kD branched PEG molecule optionally linked to the N-terminus of hIL10A via a polypeptide linker or chemical linker. (b) The "-" is a peptide bond, and (c)[hIL10A-hIL10B] is, This polypeptide is selected from the group consisting of TIFF2026521391000056.tif89150.

[0254] In some cases, the fused hIL10 polypeptides of this disclosure have an N-terminal glutamine ("1Q") residue. The N-terminal glutamine residue has been observed to spontaneously cyclize under physiological or near-physiological conditions to form pyroglutamic acid (pE). (See, e.g., Liu, et al (2011) J. Biol. Chem. 286(13):11211-11217). In some embodiments, the formation of pyroglutamic acid prevents N-terminal PEG conjugation, particularly when aldehyde chemistry is used for N-terminal PEGylation. Consequently, when PEGylating the IL-10 agonist compounds of this disclosure, particularly when aldehyde chemistry is used, IL-10 agonist compounds having an amino acid (e.g., 1Q) at position 1 are either substituted with an alternative amino acid at position 1 or deleted at position 1 (e.g., des-1Q). In some embodiments, the IL-10 agonist compounds of the present disclosure include amino acid substitutions selected from groups Q1E and Q1D.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] PEG conjugated to a polypeptide sequence may be linear or branched. Branched PEG derivatives, "star-PEG," and multi-armed PEGs 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 This includes PEG-aldehydes (e.g., Sunbright® ME-300AL) and linear 30kDa PEG-NHS esters.

[0262] In some embodiments, a linker can be used to link the fused hIL10 polypeptide to 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.

[0263] In some embodiments, the Disclosure provides a PEGylated fusion hIL10 polypeptide in which PEG is conjugated to the fusion hIL10 polypeptide, 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 embodiment of the Disclosure, PEG is a 40kD branched PEG containing two 20kD arms.

[0264] Fatty acid carriers In some embodiments, fusion hIL10 polypeptides that have an extended duration of action in mammalian subjects and are useful for implementing the present disclosure are realized by covalent bonding of the fusion hIL10 polypeptide 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 fusion hIL10 polypeptide is 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 fused hIL10 polypeptide can be acetylated at one or more lysine residues by enzymatic reaction with, for example, lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325(5942):834L2 ortho840.

[0265] nucleic acid sequence In some embodiments, the fusion hIL10 polypeptide is produced by a recombinant method using a nucleic acid sequence encoding the fusion hIL10 polypeptide. The nucleic acid sequence encoding the desired fusion hIL10 polypeptide can be synthesized by chemical means using an oligonucleotide synthesizer.

[0266] 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.

[0267] Nucleic acid molecules encoding fused hIL10 polypeptides may contain sequences that are naturally occurring or sequences that are different from naturally occurring sequences but 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).

[0268] Nucleic acid sequences can be obtained from various commercial suppliers that provide custom-made nucleic acid sequences. The amino acid sequence variants of the fusion hIL10 polypeptide of this disclosure are 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.

[0269] Methods for constructing DNA sequences and expressing them 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 can also be produced using standard recombination techniques. In the case of deletions or additions, the nucleic acid molecule is optionally digested with an appropriate restriction endonuclease. The resulting fragment can be expressed directly or further manipulated, for example, by ligating it into a second fragment. Ligation can be promoted 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.

[0270] The fusion hIL10 polypeptides of this disclosure may be synthesized directly or recombinantly 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 mature fusion hIL10 polypeptide. 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 the vector or part of a coding sequence inserted into the vector. The selected heterologous signal sequence is preferably 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 molecule. 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 molecule 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 signal peptide. In some embodiments, the signal peptide is the amino acid sequence of a human IL10 polypeptide. Includes TIFF2026521391000057.tif4128. In some embodiments, the signal peptide is the amino acid sequence of the mouse IL10 polypeptide. Includes TIFF2026521391000058.tif4128.

[0271] When the expressed fusion hIL10 polypeptide is expressed as a chimeric protein (e.g., a fusion protein containing the fusion hIL10 polypeptide and a heterologous polypeptide sequence), the chimeric protein may be encoded by a hybrid nucleic acid molecule containing a first sequence encoding all or part of the fusion hIL10 polypeptide and a second sequence encoding all or part of the heterologous polypeptide. For example, the molecule may be fused to a hexa- / octahistidine tag (SEQ ID NO: 146) 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, it should not be understood as being limited to the orientation of the elements of the fusion protein, the heterologous polypeptide may be ligated to either the N-terminus and / or C-terminus of the molecule. For example, the N-terminus may be ligated to a targeting domain, and the C-terminus may be ligated to a hexa-histidine tag (SEQ ID NO: 147) purification handle.

[0272] A reverse-translated gene can be constructed using the complete amino acid sequence of the expressed polypeptide (or fusion / chimera). DNA oligomers containing the nucleotide sequence encoding the fusion hIL10 polypeptide 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.

[0273] Codon optimization In some aspects, nucleic acid sequences encoding amino acid molecules can be “codon-optimized” to enhance their 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.

[0274] Recombinant production In some embodiments, the fused hIL10 polypeptide of this disclosure is produced by recombinant DNA technology. In a typical implementation of polypeptide recombinant 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 from the cell culture medium if a secretory leader sequence (signal peptide) is incorporated into the polypeptide.

[0275] In certain embodiments, the fusion hIL10 polypeptide may contain amino acid substitutions that provide enhanced recombinant expression compared to the expression of wild-type hIL10 without such substitutions. In some embodiments, the hIL10 mutein in the fusion hIL10 polypeptide, whose expression is increased in transfected or recombinant cells, contains an amino acid substitution at the position corresponding to residue H14 in SEQ ID NO:4. In some embodiments, the amino acid substitution at the position corresponding to H14 in SEQ ID NO:4 is selected from the group consisting of H14A, H14D, H14E, H14I, H14K, H14L, H14M, H14N, H14Q, H14R, H14S, H14T, H14Y, and H14V. In some embodiments, the amino acid substitution at the position corresponding to H14 in SEQ ID NO:4 is selected from H14D, H14C, H14G, H14P, H14F, and H14W. In some embodiments, amino acid substitutions at the position corresponding to H14 in SEQ ID NO:4 result in a substantial increase in yield while preserving STAT3 signaling.

[0276] Expression vector After construction (by synthesis, site-directed mutagenesis, or other methods), nucleic acid sequences encoding amino acid molecules can be inserted into expression vectors. Various expression vectors are available for use in different host cells and are typically selected based on the host cell for expression. Expression vectors typically contain, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Vectors include viral vectors, plasmid vectors, and integration vectors. Plasmids are an example of non-viral vectors.

[0277] In some embodiments, the vector may include sequences functionally ligated to expression regulatory elements (e.g., promoters). To facilitate the efficient expression of recombinant polypeptides, the nucleic acid sequence encoding the polypeptide sequence to be expressed is functionally ligated to transcriptional and translational regulatory sequences that are functional within a selected expression host.

[0278] 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.

[0279] Regulatory control array Expression vectors may contain regulatory sequences that are recognized by a host organism and functionally linked to a nucleic acid sequence encoding an amino acid molecule. 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. In the selection of an expression regulatory sequence, various factors understood by those skilled in the art should be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the amino acid molecule, particularly with respect to its potential secondary structure.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] host cell This disclosure further provides prokaryotic or eukaryotic cells containing and expressing one or more nucleic acid molecules encoding a fusion hIL10 polypeptide. The cells of this disclosure are transfected cells, i.e., cells into which nucleic acid molecules, such as those encoding a fusion hIL10 polypeptide, have been introduced by recombinant DNA technology.

[0287] In some embodiments, recombinant modified cells include a vector, which contains a nucleic acid sequence encoding an amino acid molecule. In some embodiments, recombinant modified cells are prokaryotic cells, such as bacterial cells. In some embodiments, recombinant modified cells are eukaryotic cells, such as mammalian cells.

[0288] 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.

[0289] In some embodiments, recombinant fusion hIL10 polypeptides or mutaines 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)).

[0290] 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.

[0291] The fused hIL10 polypeptide can be produced in prokaryotic hosts, such as the bacterium Escherichia coli, or in eukaryotic hosts, such as 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).

[0292] When wild-type hIL10 is endogenously expressed in mammalian cells, it is efficiently cleaved within the mammalian cells and expressed as a preprotein containing a signal peptide, resulting in the N-terminal amino acid of the mature wild-type hIL10 polypeptide becoming a serine residue (Ser1). As an alternative to recombinant expression in mammalian cells, fusion hIL10 polypeptides can also be recombinantly expressed in bacterial cells. Direct expression of fusion hIL10 polypeptides (i.e., not as an N-terminal fusion protein) results in the addition of an N-terminal methionine residue (i.e., an N-terminal sequence beginning with Met-Ser-Pro-…) to the fusion hIL10 polypeptide. If the Ser1 characteristic of the native N-terminal sequence of the wild-type IL10 sequence is retained at the N-terminus of hIL10A in the fusion (hIL10A-hIL10B) hIL10 polypeptide, this results in a proline (P) residue at position +2 compared to the N-terminal methionine of the fusion hIL10 polypeptide. When proline is located at position +2 relative to the N-terminal methionine of the polypeptide, endogenous bacterial methionylaminopeptidase (MAP) in bacterial host cells often fails to efficiently cleave the N-terminal methionine. (See, for example, Figure 4B in Frottin, et al. (2019) The Proteomics of N-terminal Methionine Cleavage, Molecular & Cellular Proteomics 5(12):2336-2349). As a result, direct bacterial expression of fusion hIL10 polypeptides containing the native N-terminal sequence may result in a mixture of fusion hIL10 polypeptide species, where one fraction has the N-terminal methionine residue and the other species lacks it. Such a mixture of fusion hIL10 polypeptide species may be difficult to resolve by typical manufacturing procedures, which can lead to increased processing, product loss, and other difficulties when attempting to conjugate molecules, e.g., targeted molecules or carrier molecules, e.g., PEG molecules, to the N-terminus of the fusion hIL10 polypeptide.However, by deleting Ser1 from the fusion hIL10 polypeptide (des-Ser1), the residue at position +2 relative to the N-terminal methionine is a glycine residue (G3), which provides efficient cleavage of the N-terminal methionine, promoting bacterial production of the fusion hIL10 polypeptide and providing a more homogeneous fusion hIL10 polypeptide product. In some embodiments, the disclosure provides a fusion hIL10 polypeptide (des-Ser1) containing a serine deletion at position 1 of the N-terminal hIL10 (hIL10A), numbered according to hIL10. Alternatively, it has been shown that deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus of the hIL10 molecule results in an hIL10 molecule that substantially retains the activity of hIL10. In some embodiments, the hIL10A of the fusion hIL10 polypeptide comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus of the hIL10A of the fusion hIL10 polypeptide.

[0293] In some embodiments, the resulting fusion hIL10 polypeptide or mutein is glycosylated or deglycosylated depending on the host organism used to produce the mutein. When bacteria are selected as the host, the produced molecule is deglycosylated. Eukaryotic cells, on the other hand, typically result in the glycosylation of the molecule.

[0294] In some embodiments, the amino acids of the fusion hIL10 polypeptide may contain glycosylation motifs, particularly N-linked glycosylation motifs 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 N-linked glycosylation motifs by modifying the sequence of the N-linked glycosylation motif 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. For example, the residues N116 / K117 / S118 in the wild-type sequence of human IL10 define a potential NXS N-linked glycosylation motif. Although glycosylation of hIL10 produced in mammals has not been observed, in some embodiments, the hIL10A and / or IL10B of the fused hIL10 polypeptide may contain one or more conserved amino acid substitutions at positions N116, K117, and / or S118 to prevent glycosylation at this presumed N-linked glycosylation motif.

[0295] 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.).

[0296] Transfection Expression constructs can be introduced into host cells to produce the fusion hIL10 polypeptide or mutain disclosed herein. Expression vectors containing nucleic acid sequences encoding amino acid molecules are 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 nonviral vectors under conditions that promote uptake of nonviral vectors. Examples of conditions that promote 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).

[0297] 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.

[0298] Recombinant protein recovery When a secretion leader sequence is used, the recombinantly produced molecule can be recovered from the culture medium as a secreted polypeptide. Alternatively, the molecule 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.

[0299] 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.

[0300] Recombinant molecules expressed by a transformed host can be purified according to any preferred method. Recombinant molecules can be isolated from inclusion bodies produced in *E. coli* or from conditioned media derived from a mammalian or yeast culture producing a given mutain, using cation exchange, gel filtration, and / or reversed-phase liquid chromatography. Substantially purified forms of recombinant molecules can be purified from the expression system using common biochemical procedures and used, for example, as therapeutic agents as described herein.

[0301] In some embodiments, if the molecule is expressed with the purification tag described above, this purification handle can be used to isolate the molecule 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. In some embodiments, the chelate peptide is a polyhistidine polypeptide sequence containing 4, 5, 6, 7, 8, 9, or 10 histidine residues. Such polyhistidine chelate peptides are also called "His tags" in the scientific literature. In some embodiments, the fusion hIL10 polypeptide is modified to include a chelate peptide at its N-terminus, and optionally, the chelate peptide is covalently bonded to the fusion hIL10 polypeptide via a GS linker. In some embodiments, the fusion hIL10 polypeptide is modified to include a chelate peptide at its C-terminus, and optionally, the chelate peptide is covalently bonded to the fusion hIL10 polypeptide via a linker.

[0302] The biological activity of the recovered molecules can be assayed for activation by any suitable method known in the art, and if the secretory leader sequence is used for expression, it can be evaluated in a substantially purified form or as part of a cell lysate or cell culture medium.

[0303] Pharmaceutical preparations In some embodiments, the fused hIL10 polypeptide (and / or nucleic acid encoding the fused hIL10 polypeptide, or recombinant cells modified to incorporate a nucleic acid sequence and express the fused hIL10 polypeptide) may be incorporated into a composition comprising a pharmaceutical composition. Such a composition typically comprises the 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 fused hIL10 polypeptide will be administered to subjects requiring treatment or prophylaxis.

[0304] 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).

[0305] 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).

[0306] 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.

[0307] 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.

[0308] 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.

[0309] Route of administration: Some embodiments of the therapeutic methods of the present disclosure involve the administration of a pharmaceutical formulation comprising a fused hIL10 polypeptide (and / or a nucleic acid encoding the fused hIL10 polypeptide, or a recombinant modified host cell expressing the fused hIL10 polypeptide) to a subject in need of treatment. The pharmaceutical formulation comprising the fused hIL10 polypeptide of the present disclosure may be administered to a subject in need of treatment or prophylaxis by a variety of routes of administration, including parenteral administration, oral routes, topical routes, or inhalation routes.

[0310] Parenteral administration: In some embodiments, the methods of the present disclosure involve parenteral administration of a pharmaceutical formulation comprising a fused hIL10 polypeptide (and / or a nucleic acid encoding the fused hIL10 polypeptide, or a recombinant modified host cell expressing the fused hIL10 polypeptide) 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. The pharmaceutical formulation for parenteral administration comprises a sterile aqueous solution (if water-soluble) or dispersion, and a sterile powder for immediate preparation of a sterile injection solution or dispersion. The parenteral preparation may be sealed in an ampoule, a disposable syringe, or a glass or plastic multi-dose vial. In one embodiment, the formulation is provided in a pre-filled syringe.

[0311] Oral administration: In some embodiments, the methods of the present disclosure involve the oral administration of a pharmaceutical formulation comprising a fused hIL10 polypeptide (and / or a nucleic acid encoding the fused hIL10 polypeptide, or a recombinant modified host cell expressing the fused hIL10 polypeptide) 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.

[0312] Inhalation preparations: In some embodiments, the methods of the present disclosure involve the inhalation administration of a pharmaceutical formulation comprising a fused hIL10 polypeptide (and / or a nucleic acid encoding the fused hIL10 polypeptide, or recombinant modified host cells expressing the fused hIL10 polypeptide) to a subject in need of treatment. In the case of inhalation administration, the fused hIL10 polypeptide 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.

[0313] Mucosal preparations and transdermal preparations: In some embodiments, the methods of the present disclosure involve mucosal or transdermal administration of a pharmaceutical formulation comprising a fused hIL10 polypeptide (and / or a nucleic acid encoding the fused hIL10 polypeptide, or recombinant modified host cells expressing the fused hIL10 polypeptide) 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.

[0314] Sustained-release formulations and depot formulations: In some aspects of the methods of this disclosure, the fused hIL10 polypeptide is administered in a formulation to a subject in need of treatment in order to provide sustained release of the fused hIL10 polypeptide. 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 fused hIL10 polypeptide 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 fused hIL10 polypeptide 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.

[0315] Administration of nucleic acids encoding fused hIL10 polypeptide: In some aspects of the methods of the present disclosure, delivery of the fused hIL10 polypeptide to a subject in need of treatment is achieved by administration of a nucleic acid encoding the fused hIL10 polypeptide. Methods for administering the nucleic acid encoding the fused hIL10 polypeptide 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 fused hIL10 polypeptide is administered to a subject by administration of a pharmaceutically acceptable formulation of a recombinant expression vector, which comprises a nucleic acid sequence encoding the fused hIL10 polypeptide, functionally linked to one or more functional regulatory sequences in a mammalian subject. In some embodiments, functional regulatory sequences may be selected within a limited range of cell types (or single cell types) to promote selective expression of the fused hIL10 polypeptide within a specific 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. The replication-deficient adenovirus vector may optionally contain a deletion in the E3 domain. In some aspects, adenoviruses are replication-competent adenoviruses.In some embodiments, adenoviruses are recombinant viruses with replication ability that have been engineered to selectively replicate within target cell types.

[0316] In some embodiments, particularly for the administration of the fusion hIL10 polypeptide to a subject, especially for the treatment of intestinal diseases or bacterial infections of the subject, the nucleic acid encoding the fusion hIL10 polypeptide may be delivered to the subject by administration of a recombinant modified bacteriophage vector encoding the fusion hIL10 polypeptide. Where used herein, the terms “prokaryotic virus,” “bacteriophage,” and “phage” are used without distinction herein 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 fusion hIL10 polypeptide on prokaryotic cells in the subject, while avoiding expression in mammalian cells. In the scientific literature, a wide variety of bacteriophages capable of selecting a broad range of bacterial cells have been widely identified and characterized. 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 fusion hIL10 polypeptide.

[0317] Administration of recombinant modified cells expressing fusion hIL10 polypeptide In some aspects of the methods of this disclosure, delivery of the fused hIL10 polypeptide to a subject in need of treatment is achieved by administering recombinant host cells modified to express the fused hIL10 polypeptide, which may be administered in the therapeutic and prophylactic applications 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.

[0318] In some embodiments, the nucleic acid sequence encoding the fusion hIL10 polypeptide (or a vector containing it) may be maintained extrachromosomally within recombinant modified host cells for administration. In other embodiments, the nucleic acid sequence encoding the fusion hIL10 polypeptide may be incorporated into the genome of the host cell to be administered 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 a wild-type or variant enzyme capable of catalyzing 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 may be used to inactivate a gene at a locus or to incorporate a transgene by homologous recombination (HR), i.e., by inducing a DNA double-strand break (DSB) at a 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).

[0319] In some embodiments, particularly for the administration of fused hIL10 polypeptide to the intestinal tract, the fused hIL10 polypeptide 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 fused hIL10 polypeptide may be administered orally, typically in an aqueous suspension, or rectally (e.g., by enema).

[0320] How to use This disclosure further provides a method for treating a subject 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 fused hIL10 polypeptide, (b) a pharmaceutically acceptable formulation containing the fused hIL10 polypeptide 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 fused hIL10 polypeptide functionally linked to one or more expression regulatory sequences, (d) a recombinant prokaryotic cell or recombinant mammalian cell genomically modified to contain a nucleic acid sequence encoding the fused hIL10 polypeptide functionally linked to one or more expression regulatory sequences, or (e) a recombinant prokaryotic cell or recombinant mammalian cell comprising a nucleic acid encoding the fused hIL10 polypeptide functionally linked to one or more expression regulatory sequences.

[0321] Inflammatory disorders and autoimmune disorders Disorders suitable for treatment with the fused hIL10 polypeptide of this disclosure (including pharmaceutically acceptable formulations containing the fused hIL10 polypeptide and / or encoding nucleic acid molecules, including recombinant viruses encoding such fused hIL10 polypeptides) 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 (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, 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).

[0322] ulcerative colitis In some embodiments, the Disclosure provides a method for treating a mammalian subject suffering from ulcerative colitis, comprising the step of administering a fused hIL10 polypeptide (or a pharmaceutical formulation comprising the fused hIL10 polypeptide) of the Disclosure, wherein the administration step provides improvement of one or more symptoms of ulcerative colitis. In one aspect, this disclosure relates to SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 119, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183 The present invention provides a method for treating a mammalian subject suffering from ulcerative colitis, comprising the step of administering a fusion hIL10 polypeptide 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 a polypeptide selected from the group consisting of 184, 185, and 186, wherein the administration step provides improvement of one or more symptoms of ulcerative colitis.

[0323] IFNγ-induced anemia: In some embodiments, the present 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 present disclosure to a subject, the administration step resulting in improvement of one or more symptoms of the 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). As illustrated in Figure 44, administration of a wild-type mIL10 substitute exacerbated IFNγ-induced anemia, but administration of the fusion dimer of this disclosure did not. In one aspect, this disclosure relates to SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 119, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183 The present invention provides a method for treating and / or preventing IFNγ-induced anemia, comprising the step of administering a fused hIL10 polypeptide 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 a polypeptide selected from the group consisting of 184, 185, and 186, wherein the administration step provides improvement of one or more symptoms of IFNγ-induced anemia.

[0324] Macrophage activation syndrome In some embodiments, the present disclosure provides a method for treating and / or preventing macrophage activation syndrome (MAS) in a subject, the method comprising the step of administering a therapeutically effective dose of the fused IL-10 dimer of the present 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. As shown in Figure 29, treatment with wild-type IL-10 induced CD163 expression in day 15 PEC, but the fused IL-10 dimer containing hIL10 mutain did not induce CD163. In some embodiments, the Disclosure provides a method for treating and / or preventing a target macrophage activation syndrome (MAS), the method comprising the step of administering a therapeutically effective dose of the fused IL10 dimer of the Disclosure to a target, wherein the administration step results in improvement of one or more symptoms of macrophage activation syndrome. In one aspect, this disclosure relates to SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 119, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183 The present invention provides a method for treating and / or preventing a target macrophage activation syndrome, comprising the step of administering a fusion hIL10 polypeptide 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 a polypeptide selected from the group consisting of 184, 185, and 186, wherein the administration step provides improvement of one or more symptoms of the macrophage activation syndrome.

[0325] Other examples of proliferative and / or differentiation disorders suitable for treatment with the fused hIL10 polypeptide of this disclosure (including pharmaceutically acceptable formulations comprising nucleic acid molecules containing the fused hIL10 polypeptide and / or encoding such a recombinant virus) include, but are not limited to, cutaneous disorders. Cutaneous disorders may involve abnormal activity of cells or groups of cells or layers in the dermis, epidermis, or subcutaneous tissue layers, or abnormalities in the dermal-epidermal junction. For example, cutaneous 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, slender layer, 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.

[0326] 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.

[0327] Compositions of the present disclosure (including pharmaceutically acceptable formulations comprising nucleic acid molecules containing a fused hIL10 polypeptide and / or encoding such a fused hIL10 polypeptide, and / or a recombinant virus encoding such a fused hIL10 polypeptide) may also be administered to patients who have (or may have) psoriasis or a psoriatic disorder. The term “psoriasis” is intended to have its medical meaning, namely, a disease that primarily affects the skin, resulting in raised, thickened, desquamating, and non-scarring lesions. The lesions are typically well-defined erythematous papules covered with shiny, folded 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, exanthematous psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or focal pustular psoriasis.

[0328] In some embodiments, the Disclosure provides a method for treating cancer, comprising the step of administering a therapeutically effective amount of the fused IL10 polypeptide of the Disclosure to a target, wherein the administration step results in improvement of one or more symptoms of the cancer. As previously stated, 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 compositions of the 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 colon cancer, colorectal cancer, pancreatic cancer, and liver cancer. In one embodiment, the Disclosure provides a method for preventing the development of cancers associated with chronic inflammation in a human subject by administering a prophylactically effective amount of a composition comprising a vector encoding the fused IL10 polypeptide of the Disclosure or recombinant cells expressing the fused IL10 polypeptide of the Disclosure to a target.

[0329] Administration The fused hIL10 polypeptides of this disclosure exhibit a broader therapeutic range compared to wild-type IL10. “Therapeutic range” refers to a dosage range of the fused hIL10 polypeptide that provides a target concentration 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 fused hIL10 polypeptide 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 fused hIL10 polypeptide (or a pharmaceutically acceptable formulation containing fused hIL10 polypeptide as an active ingredient), 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.

[0330] 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 z-fusion hIL10 polypeptide (or a pharmaceutically acceptable formulation containing z-fusion hIL10 polypeptide as an active ingredient) 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 fusion hIL10 polypeptide (or a pharmaceutically acceptable formulation containing fusion hIL10 polypeptide as an active ingredient), wherein the therapeutically effective dose is (a) STAT3 EC in activated human monocytes 20 In hyper- or activated human monocytes, STAT3 EC 30In 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 10 The dose is for providing a concentration less than 0.50, or less than STAT3 EC5 in activated human T cells. In some embodiments, the disclosure provides a method for treating a subject suffering from a disease disorder or condition by administering a therapeutically effective dose of fused hIL10 polypeptide (or a pharmaceutically acceptable formulation containing fused hIL10 polypeptide), wherein the therapeutically effective dose is less than STAT3 EC5 in activated human monocytes. 20 However, in activated human T cells, STAT3 EC 30 Less than STAT3 EC in activated human monocytes 50 However, in activated human T cells, STAT3 EC 20 Less than STAT3 EC in activated human monocytes 70 However, in activated human T cells, STAT3 EC 20 Less than STAT3 EC in activated human monocytes 80 However, in activated human T cells, STAT3 EC 20 It is less than, or in activated human monocytes it is greater than STAT3 Emax, but in activated human T cells it is greater than STAT3 EC 30 Less than or equal to STAT3 EC in...

Claims

1. The following formula: (hIL10A)-L n -(hIL10B) A fused human IL10 (hIL10) polypeptide comprising the polypeptide, During the ceremony, (a) hIL10A and hIL10B are human IL10 (hIL10) sequences independently selected from the group consisting of wild-type hIL10 (SEQ ID NO:4) and hIL10 mutain, wherein the hIL10 mutain independently contains one or more amino acid substitutions at positions corresponding to residues T100, H14, N18, N21, M22, R24, D25, D28, R32, E74, H90, N92, S93, E96 and R104 of SEQ ID NO:4, and optionally hIL10A has amino-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues compared to SEQ ID NO:4, and / or hIL10B has amino-terminal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues compared to SEQ ID NO:4, (b) L is an amino acid linker with a length of 1 to 30 amino acids, and (c) n = 0 (does not exist) or 1 (exists) The aforementioned polypeptide.

2. (a) The amino acid substitution at position T100 is selected from the group consisting of T100L, T100D, T100V, T100E, T100A, T100R, T100N, T100Q, T100E, T100I, T100K, T100M, and T100S. (b) The amino acid substitution at position H14 is selected from the group consisting of H14C, H14F, H14P, H14W, H14G, H14A, H14D, H14E, H14I, H14K, H14L, H14M, H14N, H14Q, H14R, H14S, H14T, H14Y, and H14V. (c) The amino acid substitution at position N18 is selected from the group consisting of N18Y, N18F, N18A, N18D, N18E, N18L, N18V, N18S, N18T, N18I, N18V, N18M, N18R, N18K, and N18H. (d) The amino acid substitution at position N21 is selected from the group consisting of N21A, N21R, N21Q, N21H, N21K, N21S, N21V, N21I, N21L, N21M, N21T, N21C, N21D, and N21E. (e) The amino acid substitution at position M22 is selected from the group consisting of M22A, M22V, M22I, M22L, M22N, M22D, M22S, M22T, M22W, and M22Q. (f) The amino acid substitution at position R24 is selected from the group consisting of R24E, R24D, R24N, R24Q, R24A, R24S, and R24T. (g) The amino acid substitution at position D25 is selected from the group consisting of D25A, D25N, D25H, D25I, D25K, D25L, D25P, D25Q, and D25V. (h) The amino acid substitution at position D28 is selected from the group consisting of D28A, D28E, D28L, D28V, D28S, D28T, D28I, D28V, D28M, D28H, D28K, and D28R. (i) The amino acid substitution at position R32 is selected from the group consisting of R32A, R32D, R32E, R32L, R32V, R32S, R32T, R32I, R32V, R32M, R32N, R32Q, R32G, R32C, R32P, R32F, R32Y, and R32H, (j) The amino acid substitution at position E74 is selected from the group consisting of E74A, E74D, E74L, E74V, E74S, E74T, E74I, E74V, E74M, E74H, E74K, and E74R. The amino acid substitution at position (k) H90 is selected from the group consisting of H90A, H90D, H90E, H90I, H90K, H90L, H90M, H90N, H90Q, H90R, H90S, H90T, H90Y, and H90V. (l) The amino acid substitution at position N92 is selected from the group consisting of N92D, N92Q, N92E, N92H, N92K, N92S, N92V, N92I, N92L, N92M, N92T, and N92A. The amino acid substitution at position (m) S93 is selected from the group consisting of S93E, S93A, S93R, S93N, S93D, S93Q, S93E, S93I, S93L, S93K, S93M, S93G, and S93V. (n) The amino acid substitution at position E96 is selected from the group consisting of E96C, E96F, E96Y, E96W, E96A, E96N, E96D, E96Q, E96H, E96K and E96S, and (o) The amino acid substitution at position R104 is selected from the group consisting of R104A, R104W, R104Y, R104F, R104H, R104D, R104E, R104N, R104Q, R104S, R104T, R104I, R104L, R104V, and R104M. The fused hIL10 polypeptide according to claim 1.

3. The fused hIL10 polypeptide according to claim 1 or 2, wherein at least one of hIL10A and hIL10B is hIL10 mutain.

4. The fused hIL10 polypeptide according to any one of claims 1 to 3, wherein both hIL10A and hIL10B are hIL10 mutaine.

5. A fused hIL10 polypeptide according to any one of claims 1 to 4, wherein hIL10A and hIL10B are the same.

6. A fused hIL10 polypeptide according to any one of claims 1 to 4, wherein hIL10A and hIL10B are different.

7. A fused hIL10 polypeptide according to any one of claims 1 to 6, wherein n = 0 (non-existent).

8. The fusion hIL10 polypeptide according to any one of claims 1 to 6, wherein n = 1 (existing) and L is a polypeptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.

9. The fused hIL10 polypeptide according to claim 8, wherein L is a GS linker.

10. GS Linker, A fused hIL10 polypeptide according to claim 9, comprising the sequence of .

11. The fusion hIL10 polypeptide according to any one of claims 1 to 10, wherein hIL10A and hIL10B are independently selected from wild-type hIL10 (SEQ ID NO: 4) or from IL10 muteins containing amino acid substitutions selected from the group consisting of N21D, N21E, N21K, M22A, M22S, M22T, M22D, M22W, R24E, D25K, E96K, E96Q, T100E, T100C, and T100L.

12. The fusion hIL10 polypeptide according to any one of claims 1 to 11, wherein hIL10A and / or hIL10B comprises the amino acid substitution T100L.

13. The fusion hIL10 polypeptide according to any one of claims 1 to 11, wherein hIL10A and / or hIL10B comprises the amino acid substitution M22A or M22S.

14. The fusion hIL10 polypeptide according to any one of claims 1 to 11, wherein hIL10A and / or hIL10B comprises the amino acid substitution E96Q.

15. The fusion hIL10 polypeptide according to any one of claims 1 to 11, wherein hIL10A and / or hIL10B comprises the amino acid substitution R24E.

16. The fusion hIL10 polypeptide according to any one of claims 1 to 11, wherein hIL10A and / or hIL10B comprises the amino acid substitution D25K.

17. The fusion hIL10 polypeptide according to any one of claims 1 to 11, wherein hIL10A and / or hIL10B comprises the amino acid substitution N21K.

18. The fusion hIL10 polypeptide according to any one of claims 1 to 11, wherein hIL10A and / or hIL10B are polypeptides having at least 90% sequence identity with a polypeptide selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, and optionally, one or both of hIL10A and hIL10B have an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues compared to the sequence of SEQ ID NO:

4.

19. The fused hIL10 polypeptide according to claim 18, wherein hIL10A and hIL10B are the same.

20. The fused hIL10 polypeptide according to claim 18, wherein hIL10A and hIL10B are different.

21. The fusion hIL10 polypeptide according to claim 19 or 20, wherein hIL10A and hIL10B each contain an amino acid substitution selected from the group consisting of N21D, N21E, N21K, M22A, M22S, M22T, M22D, M22W, R24E, D25K, E96K, E96Q, T100E, T100C, and T100L.

22. The fused hIL10 polypeptide according to claim 19 or 20, wherein hIL10A is a polypeptide selected from the group consisting of SEQ ID NO: 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 and 205, and hIL10B is a polypeptide selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155 and 156.

23. The fusion hIL10 polypeptide according to claim 21, wherein both hIL10A and hIL10B are hIL10 mutaines containing the amino acid substitution T100L.

24. The fusion hIL10 polypeptide according to claim 23, wherein both hIL10A and hIL10B are hIL10 mutaines containing the amino acid substitution T100L, hIL10A is a polypeptide having 100% sequence identity with SEQ ID NO:204, and hIL10B is a polypeptide having 100% sequence identity with SEQ ID NO:

15.

25. SEQ ID NO:24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 119 , 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 The fusion hIL10 polypeptide according to claim 21, comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a polypeptide selected from the group consisting of 179, 180, 181, 182, 183, 184, 185, and 186.

26. The fusion hIL10 polypeptide according to claim 21, comprising an amino acid sequence having 100% sequence identity with a polypeptide selected from the group consisting of SEQ ID NO: 44, 123, 124, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, and 186.

27. The fusion hIL10 polypeptide according to claim 23, comprising an amino acid sequence having at least 90% sequence identity with a polypeptide selected from the group consisting of SEQ ID NO:44, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:170, and SEQ ID NO:

185.

28. The fusion hIL10 polypeptide according to claim 27, comprising a polypeptide having 100% identity with the amino acid sequence of SEQ ID NO:

44.

29. The fusion hIL10 polypeptide according to claim 27, comprising a polypeptide having 100% identity with the amino acid sequence of SEQ ID NO:

185.

30. The fused hIL10 polypeptide according to any one of claims 1 to 29, wherein the fused hIL10 polypeptide is modified to extend its half-life in vivo.

31. The fused hIL10 polypeptide according to claim 30, wherein the modification for extending the half-life in vivo is selected from the group consisting of PEGylation, acylation, albumination, or conjugation to an Fc polypeptide.

32. The fusion hIL10 polypeptide according to claim 31, wherein the modification for extending the half-life in vivo is acylation.

33. The fused hIL10 polypeptide according to claim 31, wherein the modification for extending the half-life in vivo is conjugation to an Fc polypeptide.

34. The fused hIL10 polypeptide according to claim 33, wherein the Fc polypeptide is an Fc domain derived from hIgG1, hIgG2, hIgG3, or hIgG4, or a variant thereof.

35. The fused hIL10 polypeptide according to claim 33 or 34, wherein the Fc polypeptide comprises a sequence modified from a wild-type Fc polypeptide sequence to reduce effector function.

36. The fusion hIL10 polypeptide according to any one of claims 33 to 35, wherein the Fc polypeptide comprises one or more amino acid substitutions or deletions to promote heterodimerization.

37. The aforementioned fused hIL10 polypeptide is given by formula #1: IL10FP-L1 a -UH1-Fc1 [1] The first polypeptide and formula #2: UH2-Fc2 [2] A heterodimer Fc molecule containing a second polypeptide, During the ceremony, hIL10FP is a fused hIL10 polypeptide of the present disclosure (e.g., formula (hIL10A)-L n - (hIL10B) is a fused hIL10 polypeptide, L1 is the linker, and a is selected independently of 0 (non-existent) or 1 (existent). UH1 and UH2 are upper hinge domains of human immunoglobulins, independently selected from the group consisting of the upper hinge domains of IgG1, IgG2, IgG3, and IgG4, respectively, and optionally contain the amino acid substitution C220S (EU numbering). Fc1 is a polypeptide comprising a lower hinge, CH2 domain, and CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and contains one or more amino acid substitutions that promote heterodimerization with Fc2. Fc2 is a polypeptide comprising a lower hinge, a CH2 domain, and a CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and contains one or more amino acid substitutions that promote heterodimerization with Fc1. Optionally, the polypeptide of formula [1] and the polypeptide of formula [2] are linked by at least one interchain disulfide bond. The fused hIL10 polypeptide according to claim 33.

38. The fused hIL10 polypeptide according to claim 31, wherein the modification for extending the half-life in vivo is PEGylation.

39. The fused hIL10 polypeptide according to claim 38, wherein PEG is a linear or branched polyethylene glycol molecule having a molecular weight of approximately 2,000 to approximately 80,000 daltons, or approximately 2,000 to approximately 70,000 daltons, or approximately 5,000 to approximately 50,000 daltons, or approximately 10,000 to approximately 50,000 daltons, or approximately 20,000 to approximately 50,000 daltons, or approximately 30,000 to approximately 50,000 daltons.

40. The fused hIL10 polypeptide according to claim 39, wherein PEG is linear.

41. The fused hIL10 polypeptide according to claim 39, wherein the PEG is branched.

42. The fusion hIL10 polypeptide according to claim 39, wherein PEG is a 40kD branched PEG molecule containing two 20kD arms.

43. The fused hIL10 polypeptide according to claim 39, wherein PEG is optionally covalently bonded to the N-terminus of the polypeptide via a linker.

44. The following equation is formed when PEG is optionally covalently bonded to the N-terminus of hIL10A via a linker: The fusion hIL10 polypeptide according to claim 39, which is a 40kD branched PEG molecule containing two 20kD arms.

45. The fused hIL10 polypeptide according to claim 44, wherein PEG is covalently bonded to the N-terminus of hIL10A via an aldehyde linker.

46. A fusion hIL10 polypeptide according to any one of claims 1 to 45, exhibiting a ratio of wild-type hIL10 activity in activated human monocytes that is greater than the ratio of wild-type hIL10 activity in activated human CD8 T cells.

47. The fusion hIL10 polypeptide according to claim 46, wherein the activity of wild-type hIL10 is the induction of intracellular STAT3 signaling.

48. The fusion hIL10 polypeptide according to any one of claims 1 to 45, wherein the fusion hIL10 polypeptide exhibits an Emax of at least 30%, optionally at least 40%, and optionally at least 50% of the activity level of wild-type hIL10 in activated human monocytes, and the activity of wild-type hIL10 in activated human monocytes is selected from the group consisting of inhibition of IL1b secretion and inhibition of TNFα secretion.

49. The fusion hIL10 polypeptide according to any one of claims 1 to 45, wherein the fusion hIL10 polypeptide exhibits an Emax of less than 30%, optionally less than 20%, and optionally less than 10% of the activity level of wild-type hIL10 in activated human CD8 T cells, and the activity of wild-type hIL10 in activated human CD8 T cells is selected from the group consisting of IFNγ secretion, granzyme A secretion, and granzyme B secretion.

50. A nucleic acid sequence encoding the fusion hIL10 polypeptide according to any one of claims 1 to 45.

51. The nucleic acid sequence according to claim 50, which is mRNA.

52. The nucleic acid sequence according to claim 50, which is DNA.

53. A vector comprising a nucleic acid sequence according to any one of claims 50 to 52, functionally linked to an expression control sequence.

54. The vector according to claim 53, which is a viral vector.

55. Cells transformed by the vector according to claim 53 or 54.

56. The cell according to claim 55, which is a mammalian cell.

57. The active ingredient is (a) a fusion hIL10 polypeptide according to any one of claims 1 to 45, (b) a nucleic acid sequence according to any one of claims 50 to 52, (c) a vector according to claim 53 or 54, or (d) a cell according to claim 55 or 56. One or more pharmaceutically acceptable solvents, carriers, stabilizers, preservatives or diluents A pharmaceutical preparation containing [the specified ingredient].

58. A method for preventing or treating a mammalian subject suffering from a disease, disorder, or condition, (a) a fused hIL10 polypeptide according to any one of claims 1 to 45, (b) a nucleic acid sequence according to any one of claims 50 to 52, (c) a vector according to claim 53 or 54, or (d) a cell according to claim 55 or 56, (e) The pharmaceutical preparation according to claim 57 The method comprising the step of administering to the subject.

59. The method according to claim 58, wherein the disease, disorder, or condition is an autoimmune disease, disorder, or condition.

60. The method according to claim 59, wherein the autoimmune disease, disorder, or condition is inflammatory bowel disease (IBD).

61. The method according to claim 60, wherein IBD is Crohn's disease or ulcerative colitis.

62. The method according to claim 58, wherein the disease, disorder, or condition is cancer.

63. The method according to claim 62, wherein the cancer is a cancer resulting from chronic inflammation.

64. The method according to claim 58, wherein the disease, disorder, or condition is macrophage activation syndrome.

65. The method according to claim 58, wherein the disease, disorder, or condition is interferon-gamma induced anemia.

66. A method for producing a fusion human IL10 (hIL10) polypeptide of formula (hIL10A)-Ln-(hIL10B), comprising the steps of: transfecting a host cell with the vector according to claim 53 or 54; culturing the cell in a culture medium under conditions that enable the expression of the nucleic acid sequence encoding the fusion hIL10 polypeptide; and isolating the fusion hIL10 polypeptide from the culture medium, the method optionally further comprising the step of purifying the fusion hIL10 polypeptide.

67. The method according to claim 66, wherein the host cell is a prokaryotic cell.

68. The method according to claim 67, wherein the prokaryotic cell is an Escherichia coli (E. coli) cell.

69. The method according to any one of claims 66 to 68, wherein hIL10A is a polypeptide selected from the group consisting of SEQ ID NO: 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 and 205, and hIL10B is a polypeptide selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 148, 149, 150, 151, 152, 153, 154, 155 and 156.

70. The method according to claim 69, wherein hIL10A is a polypeptide selected from the group consisting of SEQ ID NO: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, and 186.

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